Method Of Corneal And Scleral Inlay Crosslinking And Preservation

ABSTRACT

A method of corneal and scleral inlay crosslinking and preservation is disclosed herein. The method includes cross-linking at least a portion of a donor cornea or a donor sclera so as to kill cellular elements in the portion of the donor cornea or the donor sclera, and make the portion of the donor cornea or the donor sclera less antigenic to a body portion of a recipient patient in which the portion of the donor cornea or the donor sclera is to be implanted; and storing the cross-linked donor cornea or the donor sclera for a long period of time prior to implanting the portion of the donor cornea or the donor sclera into the body portion of the recipient patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 62/780,404, entitled “Method Of Corneal And ScleralInlay Crosslinking And Preservation”, filed on Dec. 17, 2018, and is acontinuation-in-part of application Ser. No. 16/451,247, entitled“Method Of Corneal Transplantation Or Corneal Inlay Implantation WithCross-Linking”, filed on Jun. 25, 2019, which claims priority to U.S.Provisional Patent Application No. 62/849,941, entitled “Method OfCorneal Transplantation Or Corneal Inlay Implantation WithCross-Linking”, filed on May 19, 2019, and Ser. No. 16/451,247 is acontinuation-in-part of application Ser. No. 16/414,713, entitled“Method Of Corneal Transplantation Or Corneal Inlay Implantation WithCross-Linking”, filed on May 16, 2019, which claims priority to U.S.Provisional Patent Application No. 62/672,161, entitled “Drug DeliveryImplant And A Method Using The Same”, filed on May 16, 2018, and Ser.No. 16/414,713 is a continuation-in-part of application Ser. No.15/941,641, entitled “Method Of Corneal Transplantation Or Corneal InlayImplantation With Cross-Linking”, filed on Mar. 30, 2018, now U.S. Pat.No. 10,314,690, which claims priority to U.S. Provisional PatentApplication No. 62/478,914, entitled “Method Of Corneal TransplantationWith Cross-Linking”, filed on Mar. 30, 2017, and Ser. No. 15/941,641 isa continuation-in-part of application Ser. No. 15/230,445, entitled“Corneal Lenslet Implantation With A Cross-Linked Cornea”, filed on Aug.7, 2016, now U.S. Pat. No. 9,937,033, which claims priority to U.S.Provisional Patent Application No. 62/360,281, entitled “Method ofAltering the Refractive Properties of an Eye”, filed on Jul. 8, 2016,and Ser. No. 15/230,445 is a continuation-in-part of application Ser.No. 14/709,801, entitled “Corneal Transplantation With A Cross-LinkedCornea”, filed on May 12, 2015, now U.S. Pat. No. 9,427,355, whichclaims priority to U.S. Provisional Patent Application No. 61/991,785,entitled “Corneal Transplantation With A Cross-Linked Cornea”, filed onMay 12, 2014, and to U.S. Provisional Patent Application No. 62/065,714,entitled “Corneal Transplantation With A Cross-Linked Cornea”, filed onOct. 19, 2014, the disclosure of each of which is hereby incorporated byreference as if set forth in their entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISK

Not Applicable.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention generally relates to a method of corneal transplantationor corneal inlay implantation with cross-linking. More particularly, theinvention relates to method of corneal transplantation or corneal inlayimplantation with cross-linking for preventing an immune response to acorneal graft and/or rejection of the corneal graft by the patient, andfor preventing vascular and/or fibrous tissue growth on, and surroundinga keratoprosthesis lens or other type of corneal implant or inlay.

2. Background

Corneal scarring is a major cause of blindness, especially in developingcountries. There are various causes for corneal scarring, which include:bacterial infections, viral infections, fungal infections, parasiticinfections, genetic corneal problems, Fuch's dystrophy, other cornealdystrophies, corneal trauma, or failed surgery. A corneal transplant isoften required if the corneal scarring is extensive, and cannot becorrected by other means. However, there can be major complicationsassociated with a corneal transplant, such as corneal graft rejectionwherein the transplanted cornea is rejected by the patient's immunesystem.

A normal emmetropic eye includes a cornea, a lens and a retina. Thecornea and lens of a normal eye cooperatively focus light entering theeye from a far point, i.e., infinity, onto the retina. However, an eyecan have a disorder known as ametropia, which is the inability of thelens and cornea to focus the far point correctly on the retina. Typicaltypes of ametropia are myopia, hypermetropia or hyperopia, andastigmatism.

A myopic eye has either an axial length that is longer than that of anormal emmetropic eye, or a cornea or lens having a refractive powerstronger than that of the cornea and lens of an emmetropic eye. Thisstronger refractive power causes the far point to be projected in frontof the retina.

Conversely, a hypermetropic or hyperopic eye has an axial length shorterthan that of a normal emmetropic eye, or a lens or cornea having arefractive power less than that of a lens and cornea of an emmetropiceye. This lesser refractive power causes the far point to be focusedbehind the retina.

An eye suffering from astigmatism has a defect in the lens or shape ofthe cornea converting an image of the point of light to a line.Therefore, an astigmatic eye is incapable of sharply focusing images onthe retina.

While laser surgical techniques, such as laser-assisted in situkeratomileusis (LASIK) and photorefractive keratectomy (PRK) are knownfor correcting refractive errors of the eye, these laser surgicaltechniques have complications, such as post-operative pain and dry eyeor scarring. Also, these laser surgical techniques cannot be safely usedon patients with corneas having certain biomechanical properties. Forexample, corneal ectasia may occur if these laser surgical techniquesare applied to patients having thin corneas (e.g., corneas withthicknesses that are less than 500 microns).

Therefore, what is needed is a method for corneal transplantation thatreduces the likelihood that the implanted cornea will be rejected by thepatient. Moreover, a method is needed for corneal transplantation thatis capable of preserving the clarity of the transplanted cornea.Furthermore, there is a need for a method of corneal transplantationthat reduces the likelihood that the transplanted cornea will be invadedby migrating cells. Also, what is needed is a method for corneal lensletimplantation for modifying the cornea to better correct ametropicconditions without removing the tissue that potentially creates a sideeffect of keratectasia. In addition, a method is needed for corneallenslet implantation that prevents a lens implant from moving aroundinside the cornea once implanted so that the lens implant remainscentered about the visual axis of the eye, and is not rejected by thehost tissue.

Further, a number of the diseases can lead to corneal opacity and lossof sight. Among these are many infectious diseases, caused by bacterial,viral, and fungal or other organisms. Furthermore, penetrating andcontusion corneal injury in children and adults can create a cornealscar, which prevents a patient from seeing. Postsurgical procedures,such as cataract surgery, and glaucoma, etc. can also damage cornealendothelial cells with loss of clarity of the corneal tissue. A largenumber of corneal diseases have a genetic predisposition, and can cloudthe cornea over a period of time.

In order to clear the visual axis for light to reach the retina, itoften requires performing a corneal transplantation. In this process, acornea from a recently deceased person is excised and transplanted inplace of the diseased cornea in the host.

Often, the first corneal transplantation is successful for about 90% ofthe cases. However, the rest of the cases require repeated cornealtransplantation to replace a rejected one. These cases and all casesthat have vascular components to the corneal cloudiness constitute thegroup of patients in whom the corneal transplant can be rejected inabout 30% of the cases. As a general rule, the more cornealtransplantations that are performed, the greater are the chances of agraft rejection.

Medical therapy, steroids, immunosuppressants, etc. are often applied tothe cornea, but in repeated cases of corneal transplantation andcomplicated cases, they have limited success with their associated sideeffects. This process can ultimately lead to corneal edema, cellularimmune response, complete corneal cloudiness and vascularization, inaddition to dry eye. In these cases, one can remove the center part ofthe opaque cornea and create a circular pocket in the remainingperipheral cornea horizontally with a knife as known in the art. Thisproduces an anterior and a posterior flap around the central opening anda remaining part of the cornea in which a prosthetic lens with a flangeis implanted. However, often these artificial lenses can be rejectedbecause of the body's immune response.

Therefore, it is apparent that a need also exists for a cornealtransplantation method with cross-linking that prevents an immuneresponse to a corneal transplant and/or rejection of the cornealtransplant by the patient, and for a corneal transplantation method withcross-linking that prevents vascular and/or fibrous tissue growth on,and surrounding a corneal implant, inlay, or keratoprosthesis lens.

BRIEF SUMMARY OF EMBODIMENTS OF THE INVENTION

Accordingly, the present invention is directed to a method of cornealtransplantation or corneal inlay implantation with cross-linking thatsubstantially obviates one or more problems resulting from thelimitations and deficiencies of the related art.

In accordance with one or more embodiments of the present invention,there is provided a method of corneal and scleral inlay crosslinking andpreservation. The method comprising the steps of: (i) cross-linking atleast a portion of a donor cornea or a donor sclera so as to killcellular elements in the portion of the donor cornea or the donorsclera, and make the portion of the donor cornea or the donor scleraless antigenic to a body portion of a recipient patient in which theportion of the donor cornea or the donor sclera is to be implanted; and(ii) storing the cross-linked donor cornea or the donor sclera for along period of time prior to implanting the portion of the donor corneaor the donor sclera into the body portion of the recipient patient.

In a further embodiment of the present invention, the step ofcross-linking the portion of the donor cornea or the donor scleracomprises the substeps of: (a) applying a photosensitizer to the portionof the donor cornea or the donor sclera so as to penetrate the tissue ofthe donor cornea or the donor sclera; and (b) irradiating the portion ofthe donor cornea or the donor sclera with ultraviolet light so as tocross-link collagen in the portion of the donor cornea or the donorsclera so as to make the portion of the donor cornea or the donor scleraless antigenic to the body portion of the recipient patient in which theportion of the donor cornea or the donor sclera is to be implanted.

In yet a further embodiment, the photosensitizer comprises riboflavinand a cell penetrating agent, and the portion of the donor cornea or thedonor sclera is irradiated by using a laser emitting the ultravioletlight.

In still a further embodiment, the cell penetrating agent comprises oneor more nanoparticles conjugated with one or more cell penetratingpeptides (CPP) and/or activatable-cell penetrating peptides (ACPP).

In yet a further embodiment, after the substep of irradiating theportion of the donor cornea or the donor sclera with ultraviolet light,the method further comprises the steps of: (iii) implanting the portionof the donor cornea into a host cornea of the recipient patient; and(iv) applying low intensity focused ultrasound to stromal cells of thehost cornea of the recipient patient so as to kill the stromal cells ofthe host cornea.

In still a further embodiment, the step of storing the cross-linkeddonor cornea or the donor sclera for a long period of time comprisesstoring the cross-linked donor cornea or the donor sclera for a timeperiod of one year or more.

In yet a further embodiment, prior to the step of cross-linking at leasta portion of the donor cornea or the donor sclera, the method comprisesthe steps of: (iii) dissecting and removing all of the soft tissue froma donor eye so to leave only the donor cornea and/or the donor sclera,the removed soft tissue of the donor eye comprising the conjunctiva,muscles, retina, choroid, iris, ciliary body, and optic nerve; and (iv)cleansing the donor cornea and/or the donor sclera in a physiologicalsolution so as to prepare the donor cornea and/or the donor sclera forcross-linking.

In still a further embodiment, the method further comprising the stepof: (iii) cutting and shaping the donor cornea using a cuttinginstrument so as to form a lens having a desired thickness, shape, anddiameter; the lens comprising a plus or minus lens, an astigmatic lens,or a combination thereof.

In yet a further embodiment, prior to the step of cross-linking at leasta portion of the donor cornea or the donor sclera, the method comprisesthe step of: (iii) exposing the donor cornea or the donor sclera to ahigh intensity ultrasonic or hydrostatic force so as to destroy thecellular elements in the portion of the donor cornea or the donorsclera. Also, in this further embodiment, after the step ofcross-linking at least a portion of the donor cornea or the donorsclera, the method comprises the step of: (iv) cleansing the donorcornea and/or the donor sclera in a saline solution to remove thereactive oxygen species and enzymes released from the destroyed cellularelements by mechanical agitation of the donor cornea or the donor scleraor by applying a low frequency ultrasound to the donor cornea or thedonor sclera.

In still a further embodiment, after the step of cross-linking at leasta portion of the donor cornea or the donor sclera, the method comprisesthe step of: (iii) implanting the portion of the donor cornea or thedonor sclera in the body of the recipient patient to replace an injuredchin, lip, nose, or cheek; or in cosmetic surgery, to augment a chin,lip, nose, or cheek.

In yet a further embodiment, the step of cross-linking the portion ofthe donor cornea or the donor sclera comprises the substeps of: (a)applying a photosensitizer comprising riboflavin to the donor cornea orthe donor sclera so as to penetrate the tissue of the donor cornea orthe donor sclera; and (b) irradiating the donor cornea or the donorsclera with ultraviolet light so as to sterilize the donor cornea or thedonor sclera.

In still a further embodiment, the recipient patient has an infectedcorneal ulcer or scleral weakness or infection, and the method furthercomprises the steps of: (iii) applying a photosensitizer conjugated withone or more antibody-coated metallic nanoparticles, one or more cellpenetrating peptides (CPP), and one or more medications to a host eye ofthe recipient patient, the one or more medications selected from thegroup consisting of antibiotics, antifungals, anti-parasites,antivirals, and combinations thereof; and (iv) irradiating the area ofthe host eye having the infected corneal ulcer or the scleral weaknessor infection with ultraviolet light so as to kill bacteria, fungi,parasites, and/or viruses while cross-linking the area of the host eyeto resist enzymatic degradation and heal the infected corneal ulcer orthe scleral weakness or infection.

In yet a further embodiment, the method further comprises the steps of:(iii) forming cavities or holes in the portion of the cross-linked donorcornea or the donor sclera using a laser so that the portion of thedonor cornea or the donor sclera is capable of being used for sustaineddrug delivery; and (iv) co-culturing the donor cornea or the donorsclera in a medium having nanoparticles conjugated with one or moremedications and cell penetrating peptides (CPP) or lipids that penetratethe tissue scaffolds formed by the cavities or holes in the portion ofthe donor cornea or the donor sclera, the portion of the donor cornea orthe donor sclera configured to be used for sustained drug delivery whenthe portion of the donor cornea or the donor sclera is implanted in thebody portion of the recipient patient.

In still a further embodiment, the nanoparticles are formed frompolylactic, polyglycolic, polycaprolactone, and/or silicon; and whereinthe one or more medications are selected from the group consisting ofanti-inflammatory agents, steroids, nonsteroidal anti-inflammatory drugs(NSAIDs), anti-proliferative agents, immunosuppressives, antibiotics,anti-glaucoma agents, Rock inhibitors, anti-integrins, Wnt inhibitors,GSK inhibitors, anti-vascular endothelial growth factors (anti-VEGFs),and combinations thereof.

In yet a further embodiment, the step of cross-linking the portion ofthe donor cornea or the donor sclera comprises the substeps of: (a)applying riboflavin to the portion of the donor cornea or the donorsclera so as to penetrate the tissue of the donor cornea or the donorsclera; and (b) irradiating the portion of the donor cornea or the donorsclera using a laser emitting ultraviolet light so as to cross-linkcollagen in the portion of the donor cornea or the donor sclera so as tomake the portion of the donor cornea or the donor sclera less antigenicto the body portion of the recipient patient in which the portion of thedonor cornea or the donor sclera is to be implanted.

In still a further embodiment, prior to the step of cross-linking theportion of the donor cornea or the donor sclera with the riboflavin andthe ultraviolet light, the method comprises the step of: (iii) usingchemical means or mechanical means to destroy the cellular elements inthe portion of the donor cornea or the donor sclera.

In yet a further embodiment, the chemical means for destroying thecellular elements in the portion of the donor cornea or the donor scleraare selected from the group consisting of ethanol, glycerol, acids,alkalis, peracetic acid, ammonium hydroxide ionic detergents, sodiumdodecyl sulfate, sodium deoxycholate non-ionic detergents, zwitterionicdetergents, Triton X-100, benzalkonium chloride, Igepal, genipin, andcombinations thereof.

In still a further embodiment, the mechanical means for destroying thecellular elements in the portion of the donor cornea or the donor scleracomprises applying low intensity focused ultrasound to the portion ofthe donor cornea or the donor sclera.

It is to be understood that the foregoing general description and thefollowing detailed description of the present invention are merelyexemplary and explanatory in nature. As such, the foregoing generaldescription and the following detailed description of the inventionshould not be construed to limit the scope of the appended claims in anysense.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The invention will now be described, by way of example, with referenceto the accompanying drawings, in which:

FIG. 1A is a partial side cross-sectional view of an eye having ascarred cornea, wherein substantially the entire thickness of the corneais scarred;

FIG. 1B is a partial side cross-sectional view of a donor corneaundergoing cross-linking;

FIG. 1C is a partial side cross-sectional view of the eye of FIG. 1A,wherein the scarred cornea is shown being removed;

FIG. 1D is a partial side cross-sectional view of the eye of FIG. 1A,wherein the cross-linked donor cornea is shown being implanted in thelocation previously occupied by the scarred cornea;

FIG. 2A is a partial side cross-sectional view of an eye having internalcorneal scar tissue;

FIG. 2B is a partial side cross-sectional view of the eye of FIG. 2A,wherein the scarred corneal tissue has been externally removed from theeye;

FIG. 2C is a partial side cross-sectional view of the eye of FIG. 2A,wherein a cross-linked donor cornea is shown being implanted in thelocation previously occupied by the scarred corneal tissue;

FIG. 3A is a partial side cross-sectional view of an eye having internalcorneal scar tissue;

FIG. 3B is a partial side cross-sectional view of the eye of FIG. 3A,wherein the scarred corneal tissue is shown being internally removedfrom the eye;

FIG. 3C is a partial side cross-sectional view of the eye of FIG. 3A,wherein a cross-linked donor cornea is shown being implanted in thelocation previously occupied by the scarred corneal tissue;

FIG. 4A is a partial side cross-sectional view of an eye having aT-shaped corneal scar and/or diseased tissue portion;

FIG. 4B is another partial side cross-sectional view of a donor corneaundergoing cross-linking;

FIG. 4C is a partial side cross-sectional view illustrating a T-shapedportion of the cross-linked donor cornea being cut out from a remainderof the donor cornea;

FIG. 4D is a partial side cross-sectional view of the eye of FIG. 4A,wherein the T-shaped scarred and/or diseased portion of corneal tissuehas been removed from the eye;

FIG. 4E is a partial side cross-sectional view of the eye of FIG. 4A,wherein the cross-linked T-shaped donor cornea portion is shown beingimplanted in the location previously occupied by the scarred and/ordiseased corneal tissue portion;

FIG. 5A illustrates an alternative configuration for the cross-linkeddonor cornea implant, wherein the donor cornea implant has a dumbbellshape;

FIG. 5B illustrates another alternative configuration for thecross-linked donor cornea implant, wherein the donor cornea implant hasa reversed or upside down T-shape;

FIG. 6A is a side cross-sectional view of a host eye prior to antransplant procedure;

FIG. 6B is another side cross-sectional view of the host eye of FIG. 6A,which illustrates a creation of a corneal pocket therein;

FIG. 6C is another side cross-sectional view of the host eye of FIG. 6A,which illustrates an implantation of the cross-linked lamellar lensletinto the host eye;

FIG. 7A is a partial side cross-sectional view of a donor cornea beingcross-linked prior to being shaped for use in a transplant procedure;

FIG. 7B is another partial side cross-sectional view of the donor corneaof FIG. 7A, which illustrates the cutting of a cross-linked lamellarlenslet from a remainder of the cross-lined donor cornea;

FIG. 7C is a side cross-sectional view of the cross-linked lamellarlenslet after it has been appropriately shaped and removed from thedonor cornea of FIGS. 7A and 7B;

FIG. 8 is a partial side cross-sectional view illustrating the formationof a two-dimensional cut into a cornea of an eye, according to anotherembodiment of the invention;

FIG. 9 is another partial side cross-sectional view of the eye of FIG.8, which illustrates the creation of a three-dimensional pocket in thecornea of the eye;

FIG. 10 is yet another partial side cross-sectional view of the eye ofFIG. 8, which illustrates the injection of a photosensitizer into thethree-dimensional pocket in the cornea of the eye;

FIG. 11A is still another partial side cross-sectional view of the eyeof FIG. 8, which illustrates the irradiation of the stromal tissuesurrounding the three-dimensional pocket of the eye using ultravioletradiation delivered from outside of the cornea;

FIG. 11B is yet another partial side cross-sectional view of the eye ofFIG. 8, which illustrates the irradiation of the stromal tissuesurrounding the three-dimensional pocket of the eye using a fiber opticdelivering ultraviolet radiation inside the three-dimensional pocket,according to an alternative embodiment of the invention;

FIG. 12 is still another partial side cross-sectional view of the eye ofFIG. 8, which illustrates a lens implant inserted into the pocket so asto change the refractive properties of the eye;

FIG. 13 is yet another partial side cross-sectional view of the eye ofFIG. 8, which illustrates the reinjection of a photosensitizer into thethree-dimensional pocket with the lens implant disposed therein so thatthe cross-linking procedure may be repeated;

FIG. 14 is still another partial side cross-sectional view of the eye ofFIG. 8, which illustrates the re-irradiation of the stromal tissuesurrounding the three-dimensional pocket of the eye during therepetition of the cross-linking procedure;

FIG. 15 is a side cross-sectional view illustrating the creation of alens implant from an organic block of polymer using a excimer laser;

FIG. 16 is a side cross-sectional view illustrating the cutting of alens implant from an organic block of polymer using a femtosecond laser;

FIG. 17 is a side cross-sectional view illustrating a lens implant thathas been formed using a three-dimensional printing technique or amolding technique;

FIG. 18 is a front view of a cornea of an eye, according to yet anotherembodiment of the invention;

FIG. 19 is another front view of the cornea of the eye of FIG. 18,wherein a square-shaped intrastromal pocket has been formed in thecornea of the eye;

FIG. 20 is yet another front view of the cornea of the eye of FIG. 18,wherein a circular three-dimensional portion of tissue having a firstdiameter has been removed from the area within the square-shapedintrastromal pocket;

FIG. 21 is still another front view of the cornea of the eye of FIG. 18,wherein a circular three-dimensional portion of tissue having seconddiameter has been removed from the area within the square-shapedintrastromal pocket, the second diameter of the circularthree-dimensional portion of tissue in FIG. 21 being larger than thefirst diameter of the circular three-dimensional portion of tissue inFIG. 20;

FIG. 22 is yet another front view of the cornea of the eye of FIG. 18,wherein a circular lens implant has been implanted in the area where thecircular three-dimensional portion of tissue has been removed, andwherein a photosensitizer is being injected into the pocket in thecornea of the eye;

FIG. 23 is still another front view of the cornea of the eye of FIG. 18,wherein the circular lens implant is shown in the area where thecircular three-dimensional portion of tissue was removed;

FIG. 24 is a front view of an eye where a corneal graft has beenimplanted in the cornea of the eye, according to still anotherembodiment of the invention;

FIG. 25 is another front view of the eye of FIG. 24, wherein theapplication of ultraviolet radiation is being illustrated following theadministration of a cross-linking agent or photosensitizer to the corneaof the eye;

FIG. 26 is a front view of an eye where a corneal graft has beenimplanted in the cornea of the eye and neovascularization has occurred,according to yet another embodiment of the invention;

FIG. 27 is another front view of the eye of FIG. 26, wherein theapplication of ultraviolet radiation is being illustrated following theadministration of a cross-linking agent or photosensitizer to the corneaof the eye;

FIG. 28 is a front view of an eye where a keratoprosthesis lens has beenimplanted in the cornea of the eye and the cornea of the eye is beingirradiated with ultraviolet radiation following the implantation,according to still another embodiment of the invention;

FIG. 29A illustrates a first exemplary shape for the drug deliveryimplant described herein, which is in the form of a rod-shaped implant;

FIG. 29B illustrates a second exemplary shape for the drug deliveryimplant described herein, which is in the form of a curved implant;

FIG. 29C illustrates a third exemplary shape for the drug deliveryimplant described herein, which is in the form of a two-partsemi-circular implant;

FIG. 29D illustrates a fourth exemplary shape for the drug deliveryimplant described herein, which is in the form of a one-partsemi-circular implant;

FIG. 30 illustrates an exemplary coated drug delivery implant, whereinthe drug delivery implant is coated with a polymer and aphotosensitizer;

FIG. 31A illustrates a first exemplary form of the drug delivery implantdescribed herein, which is in the form of a solid tubular implant;

FIG. 31B illustrates a second exemplary form of the drug deliveryimplant described herein, which is in the form of a porous tubularimplant;

FIG. 31C illustrates a third exemplary form of the drug delivery implantdescribed herein, which is in the form of a tubular implant with openends;

FIG. 32 illustrates another exemplary form of the drug delivery implantdescribed herein, wherein the implant is tubular-shaped with holesformed in the side thereof;

FIG. 33 illustrates yet another exemplary form of the drug deliveryimplant that is similar to that which is depicted in FIG. 32, exceptthat the tubular-shaped implant of FIG. 33 has larger-sized holes formedin the side thereof;

FIG. 34 illustrates still another exemplary form of the drug deliveryimplant described herein, wherein the implant is in the form of arectangular flat tube;

FIG. 35 illustrates yet another exemplary form of the drug deliveryimplant described herein, wherein the implant is in the form of asemi-solid or silicone tubular implant with one closed end and one openend;

FIG. 36 illustrates still another exemplary form of the drug deliveryimplant described herein, wherein the implant is in the form of arectangular tube that is refillable by injection;

FIG. 37 illustrates yet another exemplary form of the drug deliveryimplant described herein, wherein the tubular implant comprises a needlefor tissue penetration and the tubular implant is capable of beingpenetrating by a needle for taking liquid biopsies;

FIG. 38A is a front view of a cornea of an eye illustrating a two-partsemi-circular drug delivery implant disposed in a cross-linked pocket inthe peripheral portion of the cornea that is spaced apart from thecentral visual axis of the eye so as not to obstruct the central portionof the eye;

FIG. 38B is a partial side cross-sectional view of the eye of FIG. 38Aillustrating the two-part semi-circular drug delivery implant disposedin the cross-linked pocket in the peripheral portion of the cornea;

FIG. 39A is a front view of a cornea of an eye illustrating a generallylinear drug delivery implant disposed in a cross-linked pocket in theperipheral portion of the cornea that is spaced apart from the centralvisual axis of the eye so as not to obstruct the central portion of theeye;

FIG. 39B is a partial side cross-sectional view of the eye of FIG. 39Aillustrating the generally linear drug delivery implant disposed in thecross-linked pocket in the peripheral portion of the cornea;

FIG. 40A is a front view of a cornea of an eye illustrating a tubulardrug delivery implant disposed in a cross-linked pocket in theperipheral portion of the cornea, wherein the implant comprises a needlefluidly coupling the implant to the anterior chamber of the eye;

FIG. 40B is a partial side cross-sectional view of the eye of FIG. 40Aillustrating the tubular drug delivery implant with the needle extendinginto the anterior chamber of the eye;

FIG. 41A is a front view of a cornea of an eye illustrating a pupil,cornea, sclera, and limbus of the eye;

FIG. 41B is a partial side cross-sectional view of the eye of FIG. 41Aillustrating an anterior chamber, iris, and lens of the eye;

FIG. 42A is a front view of a cornea of an eye illustrating a one-partsemi-circular drug delivery implant disposed in a cross-linked pocket inthe peripheral portion of the cornea;

FIG. 42B is a partial side cross-sectional view of the eye of FIG. 42Aillustrating the one-part semi-circular drug delivery implant disposedin the cross-linked pocket in the peripheral portion of the cornea;

FIG. 43A is a front view of a cornea of an eye illustrating adoughnut-shaped drug delivery implant disposed in a cross-linked pocketin the peripheral portion of the cornea;

FIG. 43B is a partial side cross-sectional view of the eye of FIG. 43Aillustrating the doughnut-shaped drug delivery implant disposed in thecross-linked pocket in the peripheral portion of the cornea;

FIG. 44A is a front view of a cornea of an eye illustrating a generallylinear drug delivery implant disposed in a cross-linked pocket in theperipheral portion of the cornea;

FIG. 44B is a partial side cross-sectional view of the eye of FIG. 44Aillustrating the generally linear drug delivery implant disposed in thecross-linked pocket in the peripheral portion of the cornea;

FIG. 45A is a front view of a cornea of an eye illustrating a drugdelivery implant disposed in a cross-linked pocket in the peripheralportion of the cornea, wherein the implant comprises a needle fluidlycoupling the implant to the anterior chamber of the eye;

FIG. 45B is a partial side cross-sectional view of the eye of FIG. 45Aillustrating the tubular drug delivery implant with the needle extendinginto the anterior chamber of the eye with the aqueous fluid of the eye;

FIG. 46 is a cutaway perspective view of an eye with a drug deliveryimplant disposed in a pocket formed in the corneal stroma of the eye;

FIG. 47 is a cross-sectional view of an eye illustrating the formationof a corneal LASIK flap in the eye;

FIG. 48 is a cutaway perspective view of the eye of FIG. 47, wherein adrug delivery implant has been placed under the corneal LASIK flap;

FIG. 49 is a cutaway perspective view of an eye illustrating theapplication of a medication to the cornea of the eye by drops;

FIG. 50 is a front view of a cornea of an eye illustrating a polymericcontact lens containing a medication disposed on the cornea of the eyeafter the eye has undergone refractive surgery or a traumatic cornealinjury;

FIG. 51 is a cross-sectional view of an eye illustrating anintracapsular drug delivery implant after the eye has undergone cataractsurgery;

FIG. 52 is a cross-sectional view of an eye illustrating a drug deliveryimplant that has been placed on the lens capsule of the eye after theeye has undergone cataract surgery;

FIG. 53 is a cross-sectional view of an eye illustrating intravitrealadministration of a medication by injecting the medication along withmicroparticles or nanoparticles;

FIG. 54A is a front view of a corneal inlay or implant;

FIG. 54B is a side view of the corneal inlay or implant of FIG. 54Aillustrating the formation of small holes in the inlay or implant madeusing a laser;

FIG. 55 is a side view of the corneal inlay or implant of FIG. 54Aillustrating the cross-linking of the inlay or implant using ultravioletradiation after the inlay or implant has been soaked in aphotosensitizer solution;

FIG. 56 is a cutaway perspective view of an eye before a corneal flaphas been formed in the eye;

FIG. 57 is another cutaway perspective view of the eye of FIG. 56illustrating the formation of a corneal flap in the eye using a laser;

FIG. 58 is yet another cutaway perspective view of the eye of FIG. 56illustrating the insertion of a corneal inlay under the flap of the eye,wherein corneal inlay is undergoing refractive correction;

FIG. 59 is still another cutaway perspective view of the eye of FIG. 56illustrating the eye after the corneal flap has been replaced;

FIG. 60 is a partial side cross-sectional view of the eye of FIG. 56illustrating the corneal inlay surrounded by the stromal tissue of thecornea;

FIG. 61A is a front view of a composite two-piece inlay or implant witha central section and a peripheral section;

FIG. 61B is a front view of another composite two-piece inlay or implantthat is divided transversely into two sections;

FIG. 61C is a front view of a composite three-piece inlay or implantthat is divided transversely into three sections;

FIG. 62 is a cutaway perspective view of an eye illustrating the corneaof the eye after the two-piece inlay or implant of FIG. 61A has beeninserted into a pocket in the cornea through a small incision;

FIG. 63 is a cutaway perspective view of an eye illustrating the corneaof the eye after the two-piece inlay or implant of FIG. 61B has beeninserted into a pocket in the cornea through a small incision;

FIG. 64 is a partial side cross-sectional view of the eye of FIGS. 62and 63 illustrating the cavity of the corneal pocket that is surroundedby stromal tissue;

FIG. 65 is a partial side cross-sectional view of the eye of FIGS. 62and 63 illustrating an inlay or implant that is disposed in the cornealpocket;

FIG. 66 is a front view of a cross-linked corneal implant or inlay;

FIG. 67 is a front view of a polymeric back sheet used for supportingthe corneal implant or inlay of FIG. 66;

FIG. 68 is a front view illustrating the corneal implant or inlay ofFIG. 66 disposed on the polymeric back sheet of FIG. 67;

FIG. 69A is a side view of a polymeric back sheet used for supporting acorneal implant or inlay that has a flat surface;

FIG. 69B is a side view of a polymeric back sheet used for supporting acorneal implant or inlay that has a convex surface;

FIG. 69C is a side view of a polymeric back sheet used for supporting acorneal implant or inlay that has a concave surface;

FIG. 70 is a front view illustrating donor corneal stromal tissue,according to one embodiment of the invention;

FIG. 71 is a cutaway perspective view of an eye before a corneal flaphas been formed in the eye;

FIG. 72 is another cutaway perspective view of the eye of FIG. 71illustrating the formation of a corneal flap in the eye;

FIG. 73 is yet another cutaway perspective view of the eye of FIG. 71illustrating the insertion of a corneal inlay under the flap of the eye,wherein corneal inlay is undergoing refractive correction;

FIG. 74 is still another cutaway perspective view of the eye of FIG. 71illustrating the application of a photosensitizer solution to thecorneal inlay by drops;

FIG. 75 is yet another cutaway perspective view of the eye of FIG. 71illustrating the cross-linking of the corneal inlay using ultravioletradiation after the photo sensitizer solution has been applied to thecorneal inlay;

FIG. 76 is a front view of an eye illustrating an intrastromal cavitythat has been created in the eye using a laser, according to anotherembodiment of the invention;

FIG. 77 is a partial side cross-sectional view of the eye of FIG. 76illustrating the intrastromal cavity;

FIG. 78 is another partial side cross-sectional view of the eye of FIG.76 illustrating a shaped donor cornea that is disposed in theintrastromal cavity;

FIG. 79 is yet another partial side cross-sectional view of the eye ofFIG. 76 illustrating the injection of a photosensitizer into the shapeddonor cornea inside the intrastromal cavity;

FIG. 80 is still another partial side cross-sectional view of the eye ofFIG. 76 illustrating the cross-linking of the shaped donor cornea usingexternally applied ultraviolet radiation; and

FIG. 81 is yet another partial side cross-sectional view of the eye ofFIG. 76 illustrating the cross-linking of the shaped donor cornea usinginternally applied ultraviolet radiation delivered using a fiber optic.

Throughout the figures, the same elements are always denoted using thesame reference characters so that, as a general rule, they will only bedescribed once.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

A first illustrative embodiment of a corneal transplant procedure with across-linked cornea is shown in FIGS. 1A-1D. The corneal transplantprocedure illustrated in FIGS. 1A-1D involves full corneal replacementof the scarred or diseased cornea by the donor cornea. In other words,FIGS. 1A-1D illustrate a penetrating keratoplasty procedure wherein thefull thickness of the scarred or diseased cornea is replaced with across-linked donor cornea (i.e., a full-thickness corneal transplant).

Referring initially to FIG. 1A, it can be seen that substantially theentire thickness of the cornea 16 of the eye 10 is scarred and/ordiseased (i.e., scarred, diseased, or scarred and diseased). FIG. 1Aalso illustrates the lens 12 and iris 14 of the eye 10, which arelocated posteriorly of the cornea 16. In this embodiment, it isnecessary to replace substantially the entire thickness of the cornea 16with a donor cornea.

In FIG. 1B, the cross-linking 18 of the clear donor cornea 20 isdiagrammatically illustrated. As depicted in FIG. 1B, only the frontportion 20 a of the donor cornea 20 is cross-linked. That is, thecross-linking does not extend all the way to the rear portion 20 b ofthe donor cornea 20. It is to be understood that the cross-linking 18 ofthe donor cornea 20 may also be done after implanting the donor corneainto the eye of the patient, rather than before implantation as shown inthe illustrative example of FIGS. 1A-1D. Also, it is to be understoodthat all or just a part of the donor cornea 20 may be cross-linked.

In the illustrative embodiments described herein (i.e., as depicted inFIGS. 1A-1D, 2A-2C, and 3A-3C), the cross-linking of the clear donorcornea may comprise the steps of: (i) applying a photosensitizer to thedonor cornea, the photosensitizer facilitating cross-linking of thedonor cornea; and (ii) irradiating the donor cornea with ultravioletlight so as to activate cross-linkers in the donor cornea and therebystrengthen the donor cornea. The photosensitizer may comprise riboflavinor a solution comprising a liquid suspension having nanoparticles ofriboflavin. The cross-linker may have between about 0.1% Riboflavin toabout 100% Riboflavin or any other suitable range or specific percentagetherein. The ultraviolet radiation or rays used to irradiate the donorcornea may be between about 370 nanometers and about 380 nanometers (orbetween 370 nanometers and 380 nanometers). The radiation is preferablyabout 3 mW or more as needed and emanates from a laser source at about a3 cm distance from the donor cornea for about 30 minutes or less. Thetime of the exposure can vary depending on the light intensity, focus,and the concentration of riboflavin. However, the ultraviolet radiationcan be applied at any suitable distance, time or wavelength. Preferably,cross-linking the donor cornea does not significantly change therefractive power of the donor cornea; however, if desired, cross-linkingcan change the refractive power of the donor cornea to any suitabledegree.

In addition to Riboflavin, other suitable cross linking agents are lowcarbon carbohydrates, such as pentose sugar (e.g., ribose) or hexosesugar (e.g., glucose), or complex carbohydrates. Other crosslinkingagents may include Transaminidases, transglutaminases or anaturally-derived cross-linker named malic acid derivative (MAD)concentrations higher than 30 mM, commercially available cross-linkerssuch as 1-ethyl-3-(3(′-dimethylaminopropyl) carbodiimide (EDC), orethyl-3(3-dimethylamino) propyl carbodiimide (EDC), etc. Thecross-linking may also be done postoperatively by the application ofother crosslinking agents, such as Triglycidylamine (TGA) synthesizedvia reacting epichlorhydrin and a carbodiimide, or the oxidized glycogenhexoses. The ribose, glucose and similar agents may penetrate the corneaeasily using drops, gel, or the slow release mechanisms, nanoparticle,microspares, liposome sets. In addition, the crosslinkers may bedelivered with Mucoadhesives.

In one or more embodiments, all or part of the donor cornea iscross-linked. Also, in one or more embodiments, a very highconcentration of Riboflavin may be used because the in vitrocross-linking process may be stopped whenever needed prior to thetransplantation of the donor cornea in the host eye. In addition, thepower of the ultraviolet (UV) laser may also be increased so as tocross-link the tissue of the donor cornea faster. The use of a highconcentration of Riboflavin, and the increasing of the ultraviolet (UV)laser power, are not possible during an in vivo cross-linking procedurebecause the aim of such an in vivo procedure is to protect the cells ofthe host cornea. Also, the in vivo process cannot be controlled asefficiently as in the vitro crosslinking of the corneal transplant.

In one or more embodiments, the donor cornea may be extracted from ahuman cadaver, or the cornea may be reconstructed as known in tissueengineering in vitro and three-dimensionally (3D) printed. Cross-linkingof a culture-grown cornea eliminates the cellular structure inside thecornea. If needed again, the healthy corneal endothelium of the patientmay be grown in vitro for these tissues by placing them on the concavesurface of the cornea and encouraging their growth under laboratorycontrol conditions prior to the transplantation.

In the embodiments where the donor cornea is tissue culture grown, thecornea may be formed from mesenchymal fibroblast stem cells, embryonicstem cells, or cells derived from epithelial stem cells extracted fromthe same patient, or a mixture of these cells. Using known tissueculture techniques, the cells may produce a transparent corneal stroma.This culture-grown corneal stroma will not have a corneal epithelium ora corneal endothelium. Thus, it eliminates the complexity of developinga full thickness cornea in the tissue culture. This stromal transplantmay be used as a lamellar or partial thickness replacement of theexisting host cornea. This transplant may also be used to augment or addto the thickness of the host cornea. This transparent corneal stroma maybe transplanted either prior to, or after being cross-linked usingvarious cross-linking methods.

In one or more embodiments, the cross-linked donor cornea may be sizedand precisely cut with a femtosecond laser to the desired shape andcurvature to replace the removed host cornea so that the refractiveerrors of the recipient are also automatically corrected with thecross-linked cornea.

Now, referring to FIG. 1C, it can be seen that the scarred and/ordiseased cornea 16 is shown being removed from the eye 10. The scarredand/or diseased cornea 16 may be removed from the eye 10 by usingvarious suitable means, such as mechanical means or cutting using alaser. When mechanical means are used to remove the scarred and/ordiseased cornea 16 from the eye 10, the scarred and/or diseased cornea16 may initially be cut away or dissected from the remainder of the eye10 using a sharp mechanical instrument (e.g., a surgical micro-knife, aneedle, a sharp spatula, a pair of micro-scissors), and thensubsequently removed or extracted with a pair of micro-forceps. Whenlaser cutting is used to remove the scarred and/or diseased cornea 16from the eye 10, the scarred and/or diseased cornea 16 may be cut awayusing a suitable laser, such as a femtosecond laser. Also, in someembodiments, the mechanical means for cutting and extraction (e.g., thesurgical micro-knife and/or pair of micro-scissors) may be used incombination with the laser means (e.g., the femtosecond laser).

In one or more embodiments, the donor cornea may be shaped and cut withthe femtosecond laser prior to the cross-linking thereof so as toreplace part or all of the recipient cornea which is cut with thefemtosecond laser. In these one or more embodiments, the entire donorand host cornea together may be cross-linked with Riboflavin and UVradiation. These procedures may also be performed on a culture-growntransplant cornea.

Then, as shown in FIG. 1D, after the scarred and/or diseased cornea 16has been removed from the eye 10, the cross-linked donor cornea 20 isimplanted into the eye 10 of the patient in the location previouslyoccupied by the scarred and/or diseased cornea 16. After implantation ofthe cross-linked donor cornea 20, sutures or a suitable adhesive may beutilized to secure the cross-linked donor cornea 20 in place on the eye10. When sutures are used for holding the donor cornea 20 in place, thesutures may comprise nylon sutures, steel sutures, or another suitabletype of non-absorbable suture. When the cornea 16 is subsequentlyablated after the implantation of the donor cornea, as will be describedhereinafter, additional sutures may be required after ablation.

In one or more embodiments, a biodegradable adhesive is used in acorneal transplantation procedure with the cross-linked donor cornea 20described above, or with a non-cross-linked corneal transplant. In theseone or more embodiments, the biodegradable adhesive obviates the needfor a suture in the corneal transplant procedure. Sutures generallydistort the surface of the cornea and can produce an opticallyunacceptable corneal surface. Also, the use of the biodegradableadhesive obviates the need for glues requiring exothermic energy. Gluesthat use an exothermic effect, such as Fibronectin, need thermal energyto activate their adhesive properties. This thermal energy, such as thatdelievered by a high-powered laser, produces sufficient heat tocoagulate the Fibronectin and the tissue that it contacts. Any thermaleffect on the cornea produces: (i) corneal opacity, (ii) tissuecontraction, and (iii) distortion of the optical surface of the cornea.The tissue adhesion created by these glues, including Fibronectin orfibrinogen, is flimsy and cannot withstand the intraocular pressure ofthe eye.

In fact, sutures are superior to these types of adhesives because thewound becomes immediately strong with sutures, thereby supporting thenormal intraocular pressure of between 18 and 35 mmHg. In contrast tothe use of a suture in which distortion that is caused by sutureplacement can be managed by cutting and removing the suture, thedistortion caused by the coagulated corneal tissue cannot be corrected.

Other glues, such as cyanoacrylate, become immediately solid aftercoming into contact with the tissue or water. These glues produce arock-hard polymer, the shape of which cannot be controlled afteradministration. Also, the surface of the polymer created by these gluesis not smooth. Thus, the eyelid will rub on this uneven surface, and theuneven surface scratches the undersurface of the eyelid when the eyelidmoves over it. In addition, the cyanoacrylate is not biodegradable orbiocompatible. As such, it causes an inflammatory response if applied tothe tissue, thereby causing undesirable cell migration andvascularization of the cornea.

Thus, by using a biocompatible and absorbable acrylate or otherbiodegradable glues that do not need exothermic energy for the processof adhesion (i.e., like fibronectin or fibrinogen), one is able tomaintain the integrity of the smooth corneal surface. In one or moreembodiments, the biocompatible and biodegradable adhesive may be paintedonly at the edges of the transplant prior to placing it in the host ordiseased cornea. In these embodiments, the biocompatible andbiodegradable adhesive only comes into contact with the host tissue atthe desired predetermined surface to create a strong adhesion. Theadhesion may last a few hours to several months depending on thecomposition of the molecule chosen and the concentration of the activecomponent.

Other suitable biodegradable adhesives or glues that may be used inconjunction with the transplant include combinations of gallic acid,gallic tannic acid, Chitosan, gelatin, polyphenyl compound, Tannic Acid(N-isopropylacrylamide (PNIPAM), and/or Poly(N-vinylpyrrolidone) withpolyethylene glycol (PEG). That is, polyethylene glycol (PEG) may bemixed with any one or plurality of gallic acid, gallic tannic acid,Chitosan, gelatin, polyphenyl compound, Tannic Acid(N-isopropylacrylamide (PNIPAM), and Poly(N-vinylpyrrolidone), so as toform a molecular glue. These adhesives are suitable for the use on thecornea because they create a tight wound that prevents leakage from thecorneal wound and maintain the normal intraocular pressure shortly aftertheir application and also do not distort the wound by causing tractionon the tissue.

In one or more embodiments, the donor cornea may be temporarily suturedto the host cornea by only a few single sutures to the host cornea.Then, the sutures may be removed immediately after donor cornea is fixedto the host cornea with a suitable adhesive.

A second illustrative embodiment of a corneal transplant procedure witha cross-linked cornea is shown in FIGS. 2A-2C. Unlike the firstembodiment described above, the corneal transplant procedure illustratedin FIGS. 2A-2C does not involve full corneal replacement of the scarredor diseased cornea by the donor cornea. Rather, FIGS. 2A-2C illustrate alamellar keratoplasty procedure wherein only a portion of the cornea 16′of the eye 10′ contains scarred and/or diseased tissue (i.e., afull-thickness corneal section is not removed). In the procedure ofFIGS. 2A-2C, an internal scarred and/or diseased portion 16 a′ of thecornea 16′ is externally removed from the eye 10′ of a patient.

Referring initially to FIG. 2A, it can be seen that only an internalportion 16 a′ of the cornea 16′ is scarred and/or diseased. As such, inthis embodiment, it is not necessary to replace the entire thickness ofthe cornea 16 with a donor cornea as was described above in conjunctionwith FIGS. 1A-1D, but rather just a portion of the cornea 16′.

Next, referring to FIG. 2B, it can be seen that the scarred and/ordiseased portion 16 a′ has been externally removed from the cornea 16′of the eye 10′ such that the cornea 16′ comprises a cavity 19 disposedtherein for receiving the donor cornea. Because an external approach wasutilized for removing the scarred and/or diseased portion 16 a′ of thecornea 16′, the cavity 19 comprises a notch-like void in the outside oranterior surface of the cornea 16′. As described above for the firstembodiment, the scarred and/or diseased corneal portion 16 a′ may beremoved from the remainder of the cornea 16′ using various suitablemeans, such as mechanical means or the laser cutting means (e.g.,femtosecond laser) described above.

Finally, as shown in FIG. 2C, after the scarred and/or diseased portion16 a′ has been removed from the remainder of the cornea 16′ of the eye10′, the cross-linked donor cornea or cross-linked donor corneal portion20′ is implanted into the eye 10′ of the patient in the locationpreviously occupied by the scarred and/or diseased corneal portion 16a′. As described above, after implantation of the cross-linked donorcorneal portion 20′ into the eye 10′, sutures or a suitable adhesive(e.g., the biocompatible and biodegradable adhesive described above) maybe utilized to secure the cross-linked donor corneal portion 20′ inplace on the host cornea of the eye 10′.

After the cross-linked donor corneal portion 20′ is implanted into theeye 10′ of the patient, a portion of the cornea 16′ may be ablated so asto change the refractive properties of the eye (e.g., to give thepatient perfect or near perfect refraction). The ablation of the portionof the cornea 16′ may be performed using a suitable laser 34, such as anexcimer laser. The ablation by the laser causes the ablated tissue toessentially evaporate into the air. Also, the ablation of the portion ofthe cornea 16′ may be done intrastromally, as with LASIK (laser-assistedin situ keratomileusis), or on the surface of the cornea, as with PRK(photorefractive keratectomy). The ablation may be performed apredetermined time period after the corneal transplantation so as toenable the wound healing process of the recipient's cornea to becompleted. It is to be understood that the ablation, which follows thecorneal transplantation, may be performed in conjunction with any of theembodiments described herein.

It is also to be understood that, in some alternative embodiments, theablation may be performed prior to the transplantation of the donorcornea, rather than after the transplantation of the donor cornea. Forexample, in one or more alternative embodiments, a lenticle may beprecisely cut in the tissue of a culture-grown stroma of a donor corneaby using a femtosecond laser so that when implanted into the hostcornea, it corrects the residual host eye's refractive error.

A third illustrative embodiment of a corneal transplant procedure with across-linked cornea is shown in FIGS. 3A-3C. Like the second embodimentdescribed above, the corneal transplant procedure illustrated in FIGS.3A-3C only involves replacing a scarred and/or diseased portion 16 a″ ofthe cornea 16″ with a donor corneal portion. Thus, similar to the secondembodiment explained above, FIGS. 3A-3C illustrate a lamellarkeratoplasty procedure wherein only a portion of the cornea 16″ of theeye 10″ contains scarred and/or diseased tissue (i.e., a full-thicknesscorneal section is not removed). Although, in the procedure of FIGS.3A-3C, an internal scarred and/or diseased portion 16 a″ of the cornea16″ is internally removed from the eye 10″ of a patient, rather thanbeing externally removed as in the second embodiment of FIGS. 2A-2C.

Referring initially to FIG. 3A, it can be seen that only an internalportion 16 a″ of the cornea 16″ of the eye 10″ is scarred and/ordiseased. As such, in this embodiment, like the preceding secondembodiment, it is not necessary to replace the entire thickness of thecornea 16″ with a donor cornea, but rather just a portion of the cornea16″.

Next, referring to FIG. 3B, it can be seen that the scarred and/ordiseased portion 16 a″ is being internally removed from the remainder ofthe cornea 16″ using a pair of forceps 22 (i.e., mechanical means ofremoval are illustrated in FIG. 3B). Advantageously, because an internalapproach is being utilized for removing the scarred and/or diseasedportion 16 a″ of the cornea 16″, the cornea 16″ will not comprise thenotch-like cavity 19 disposed in the outside or anterior surface of thecornea, which was described in conjunction with the preceding secondembodiment. As described above for the first and second embodiments, thescarred and/or diseased corneal portion 16 a″ may be removed from theremainder of the cornea 16″ using other suitable alternative means, suchas laser cutting techniques (e.g., using a femtosecond laser).Advantageously, the femtosecond laser is capable of cutting inside thetissue without involving the surface of the tissue. The cut part of thetissue can then be removed by other means (e.g., micro-forceps).

Finally, as shown in FIG. 3C, after the scarred and/or diseased cornealportion 16 a″ has been removed from the remainder of the cornea 16″ ofthe eye 10″, the cross-linked donor cornea or cross-linked donor cornealportion 20″ is implanted into the eye 10″ of the patient in the locationpreviously occupied by the scarred and/or diseased corneal portion 16a″. After implantation of the cross-linked donor corneal portion 20″,sutures or a suitable adhesive (e.g., the biocompatible andbiodegradable adhesive described above) may be utilized to secure thecross-linked donor corneal portion 20″ in place on the host cornea ofthe eye 10″. Advantageously, the cross-linked donor corneal portion 20″,which is strengthened by the cross-linking performed thereon, reinforcesthe cornea 16″ and greatly reduces the likelihood of corneal graftrejection.

It is to be understood that the scarred and/or diseased corneal portion16 a″ that is removed from the cornea 16″ may also be replaced withstroma stem cells or mesenchymal stem cells, which can be contained in amedium, and then injected in the internal cavity previously occupied bythe scarred and/or diseased corneal tissue 16 a″.

In one or more embodiments, mesenchymal stem cells also may be injectedinside the donor cornea before or after transplantation. In addition, inone or more embodiments, daily drops of a Rho Kinase inhibitor may beadded to the host eye after the surgery. The use of a medication, suchas a Rho Kinase inhibitor, with the stem cells will encourage stem cellproliferation.

A fourth illustrative embodiment of a corneal transplant procedure witha cross-linked cornea is shown in FIGS. 4A-4E. Like the second and thirdembodiments described above, the corneal transplant procedureillustrated in FIGS. 4A-4E only involves replacing a scarred and/ordiseased portion 16 a″′ of the cornea 16″′ with a donor corneal portion.Thus, similar to the second and third embodiments explained above, FIGS.4A-4E illustrate a lamellar keratoplasty procedure wherein only aportion of the cornea 16″′ of the eye 10″′ contains scarred and/ordiseased tissue (i.e., a full-thickness corneal section is not removed).Although, in the procedure of FIGS. 4A-4E, a different-shaped scarredand/or diseased portion 16 a′″ of the cornea 16′″ is removed.

Referring initially to FIG. 4A, it can be seen that only a portion 16a″′ of the cornea 16″′ having a T-shape or “top hut” shape is scarredand/or diseased. As such, in this embodiment, it is not necessary toreplace the entire thickness of the cornea 16″′ with a donor cornea aswas described above in conjunction with FIGS. 1A-1D, but rather just aportion 16 a″′ of the cornea 16″′. In this illustrative embodiment, theback side of the cornea 16″′ is maintained (see e.g., FIG. 4D).

In FIG. 4B, the cross-linking 18′ of the clear donor cornea 20′ isdiagrammatically illustrated. As mentioned above, it is to be understoodthat all or just a part of the donor cornea 20′ may be cross-linked.Then, in FIG. 4C, it can be seen that a portion 20 a′ of the clear donorcornea 20′, which has a T-shape or “top hut” shape that matches theshape of the scarred and/or diseased portion 16 a″′ of the cornea 16″′,is cut out from the remainder of the clear donor cornea 20′ such that ithas the necessary shape. In one or more embodiments, the portion 20 a′may be cut from the clear donor cornea 20′ and appropriately shapedusing a femtosecond laser. As shown in FIGS. 5A and 5B, other suitablyshaped cross-linked corneal portions may be cut from the clear donorcornea 20′, such as a dumbbell-shaped corneal portion 20 a″ (see FIG.5A) or a corneal portion 20 a″′ having a reversed T-shape or “reversedtop hut” shape (see FIG. 5B), in order to accommodate correspondinglyshaped scarred and/or diseased areas in the host cornea.

Next, referring to FIG. 4D, it can be seen that the scarred and/ordiseased portion 16 a″′ having the T-shape or “top hut” shape has beenremoved from the cornea 16″′ of the eye 10″′ such that the cornea 16″′comprises a cavity 19′ disposed therein for receiving the donor cornea.As described above for the first three embodiments, the scarred and/ordiseased corneal portion 16 a″′ may be removed from the remainder of thecornea 16″′ using various suitable means, such as mechanical means orthe laser cutting means (e.g., femtosecond laser) described above.

Finally, as shown in FIG. 4E, after the scarred and/or diseased portion16 a′″ has been removed from the remainder of the cornea 16″′ of the eye10′″, the cross-linked donor corneal portion 20 a′ is implanted into theeye 10″′ of the patient in the location previously occupied by thescarred and/or diseased corneal portion 16 a′″. Because the shape of thetransplant corresponds to that of the removed portion 16 a″′ of thecornea 16″′, the transplant sits comfortably in its position in the hostcornea. As described above, after implantation of the cross-linked donorcorneal portion 20 a′ into the eye 10′″, sutures or a suitable adhesive(e.g., the biocompatible and biodegradable adhesive described above) maybe utilized to secure the cross-linked donor corneal portion 20 a′ inplace on the host cornea 16′″ of the eye 10″′. For example, if abiocompatible and biodegradable adhesive is used to secure thecross-linked donor corneal portion 20 a′ in place in the cornea 16″′ ofthe eye 10″′, the edges of the donor corneal portion 20 a′ are coatedwith the biocompatible and biodegradable adhesive so as to give thetransplant a reliable stability. In this case, it is desirable to havethe attachment of the transplant maintained by the biocompatible andbiodegradable adhesive for a period of months (i.e., it is desirable forthe transplant to be secured in place by the biocompatible andbiodegradable adhesive for as long as possible).

An illustrative embodiment of a corneal lenslet implantation procedurewith a cross-linked cornea is shown in FIGS. 6A-6C and 7A-7C. Similar tothe second, third, and fourth embodiments described above, FIGS. 6A-6Cand 7A-7C illustrate a lamellar keratoplasty procedure wherein only aportion of the cornea 16″″ of the host eye 10″″ is removed during theprocedure (i.e., a full-thickness corneal section is not removed).Although, the procedure of FIGS. 6A-6C and 7A-7C differs in severalimportant respects from the abovedescribed procedures. In thisembodiment, the corneal transplant is cross-linked in vitro. Then, usinga femtosecond laser or an excimer laser, the surgeon carves out orablates a three-dimensional (3D) corneal cross-linked augment from thedonor cornea 20″′ that exactly compensates for the refractive error ofthe recipient of the transplant. That is, the corneal cross-linkedaugment or inlay may be cut to the desired shape using a femtosecondlaser, or the inlay may be shaped in vitro using an excimer laser priorto its implantation in the cornea 16″″ of the host eye 10″″. Aftermaking an internal pocket 28 in the recipient cornea 16″″ of the hosteye 10″″ with a femtosecond laser, the cross-linked transplant is foldedand implanted in a predetermined fashion inside the host's cornealpocket 28 to provide stability to the eye 10″″ having keratoconus,keratoglobus, a thin cornea or abnormal corneal curvature, therebypreventing future corneal ectasia in this eye 10″″ and correcting itsrefractive errors. Advantageously, the procedure of this embodimentcomprises a lamellar cross-linked corneal transplantation, whichadditionally results in simultaneous correction of the refractive errorof the eye 10″″ of the patient. As used herein, the term “lenslet”refers to a lens implant configured to be implanted in a cornea of aneye. The lens implant may be formed from an organic material, asynthetic material, or a combination of organic and synthetic materials.

Now, with reference to FIGS. 6A-6C and 7A-7C, the illustrativeembodiment will be described in further detail. The host eye 10″″ withlens 12′, cornea 16″″, and optic nerve 24 is shown in FIG. 6A, while thedonor cornea 20′″ is depicted in FIG. 7A. The donor cornea 20′″ of FIG.7A may be a cross-linked cornea of a cadaver or a tissue culture-growncornea that has been cross-linked. Turning to FIG. 6B, it can be seenthat an internal corneal pocket 28 is created in the cornea 16″″ of thehost eye 10″″ (e.g., by using a suitable laser, which is indicateddiagrammatically in FIG. 6B by lines 30).

In FIG. 7A, the cross-linking 18″ of the donor cornea 20′″ isdiagrammatically illustrated. As mentioned in the preceding embodiments,it is to be understood that all or just a part of the donor cornea 20″′may be cross-linked. Then, after the donor cornea 20′″ of FIG. 7A hasbeen cross-linked (e.g., by using a photosensitizer in the form ofriboflavin and UV radiation as described above), it can be seen that across-linked lamellar lenslet 26 is cut out from the remainder of thedonor cornea 20″′ (e.g., by using a suitable laser, which is indicateddiagrammatically in FIG. 7B by lines 32) such that it has the necessaryshape for implantation into the host eye 10″″. As explained above, thecross-linked lamellar lenslet 26 may be cut from the donor cornea 20″′and appropriately shaped using a femtosecond laser or an excimer laser.The cross-linked lamellar lenslet 26 is capable of being prepared to anyrequisite shape using either the femtosecond laser or the excimer laser.FIG. 7C illustrates the shaped cross-linked lamellar lenslet 26 after ithas been removed from the remainder of the donor cornea 20″′.

Finally, as shown in FIG. 6C, the cross-linked lamellar lenslet 26 isimplanted into the cornea 16″″ of the host eye 10″″ of the patient inthe location where the pocket 28 was previously formed. Because theshape of the transplant corresponds to that of the pocket 28 formed inthe eye 10″″, the transplant sits comfortably in its position in thehost cornea 16″″. As described above, after implantation of thecross-linked lamellar lenslet 26 into the eye 10″″, the refractiveerrors of the eye 10″″ have been corrected because the cross-linkedlamellar lenslet 26 has been appropriately shaped to compensate for thespecific refractive errors of the host eye 10″″ prior to itsimplantation into the eye 10″″. In addition, as explained above, theimplantation of the cross-linked lamellar lenslet 26 provides additionalstability to an eye having keratoconus, keratoglobus, a thin cornea, orabnormal corneal curvature.

Another illustrative embodiment of a corneal lenslet implantationprocedure with a cross-linked cornea is shown in FIGS. 8-14. In general,the procedure illustrated in these figures involves forming atwo-dimensional cut into a cornea of an eye; creating athree-dimensional pocket in the cornea of the eye, cross-linking theinterior stroma, and inserting a lenslet or lens implant into thethree-dimensional pocket after the internal stromal tissue has beencross-linked.

Initially, in FIG. 8, the forming of a two-dimensional cut 115 into thecornea 112 of the eye 110 is diagrammatically illustrated. Inparticular, as shown in the illustrative embodiment of FIG. 8, thetwo-dimensional cut 115 is formed by making an intrastromal incision inthe cornea 112 of the eye 110 using a femtosecond laser (i.e., theincision is cut in the cornea 112 using the laser beam(s) 114 emittedfrom the femtosecond laser). Alternatively, the two-dimensional cut 115may be formed in the cornea 112 of the eye 110 using a knife.

Then, in FIG. 9, the forming of a three-dimensional corneal pocket 116in the cornea 112 of the eye 110 is diagrammatically illustrated. Inparticular, as shown in the illustrative embodiment of FIG. 9, thethree-dimensional corneal pocket 116 is formed by using a spatula 118.The formation of the intracorneal pocket 116 in the cornea 112 of theeye 110 allows one to gain access to the tissue surrounding the pocket116 (i.e., the interior stromal tissue surrounding the pocket 116).

Turning again to FIGS. 8 and 9, in the illustrative embodiment, thecorneal pocket 116 formed in the cornea 112 of the eye 110 may be in theform of an intrastromal corneal pocket cut into the corneal stroma. Afemtosecond laser may be used to form a 2-dimensional cut into thecornea 112, which is then opened with a spatula 118 to create a3-dimensional pocket 116. In one embodiment, a piece of the cornea 112or a cornea which has a scar tissue is first cut with the femtosecondlaser. Then, the cavity is cross-linked before filling it with animplant or inlay 128 to replace the lost tissue with a clear flexibleinlay or implant 128 (see FIG. 12).

In one embodiment, a three-dimensional (3D) uniform circular, oval, orsquared-shaped corneal pocket 116 is cut with a femtosecond laser andthe tissue inside the pocket is removed to produce a three-dimensional(3D) pocket 116 to be cross-linked with riboflavin and implanted with aprepared implant.

After the pocket 116 is formed using the spatula 118, a photosensitizeris applied inside the three-dimensional pocket 116 so that thephotosensitizer permeates the tissue surrounding the pocket 116 (seeFIG. 10). The photosensitizer facilitates the cross-linking of thetissue surrounding the pocket 116. In the illustrative embodiment, thephotosensitizer is injected with a needle 120 inside the stromal pocket116 without lifting the anterior corneal stroma so as to cover theinternal surface of the corneal pocket 116. In one or more embodiments,the photosensitizer or cross-linker that is injected through the needle120 inside the stromal pocket comprises riboflavin, and/or a liquidsuspension having nanoparticles of riboflavin disposed therein.Preferably, the cross-linker has between about 0.1% riboflavin to about100% riboflavin therein (or between 0.1% and 100% riboflavin therein).Also, in one or more embodiments, an excess portion of thephotosensitizer in the pocket 116 may be aspirated through the needle120 until all, or substantially all, of the excess portion of thephotosensitizer is removed from the pocket 116 (i.e., the excesscross-linker may be aspirated through the same needle so that the pocket116 may be completely emptied or substantially emptied).

Next, turning to the illustrative embodiment of FIG. 11A, shortly afterthe photosensitizer is applied inside the pocket 116, the cornea 112 ofthe eye 110 is irradiated from the outside using ultraviolet (UV)radiation 122 so as to activate cross-linkers in the portion of thetissue surrounding the three-dimensional pocket 116, and thereby stiffenthe cornea 112, prevent corneal ectasia of the cornea 112, and killcells in the portion of the tissue surrounding the pocket 116. In theillustrative embodiment, the ultraviolet light used to irradiate thecornea 112 may have a wavelength between about 370 nanometers and about380 nanometers (or between 370 nanometers and 380 nanometers). Also, inthe illustrative embodiment, only a predetermined anterior stromalportion 124 of the cornea 112 to which the photosensitizer was appliedis cross-linked (i.e., the surrounding wall of the corneal pocket 116),thereby leaving an anterior portion of the cornea 112 and a posteriorstromal portion of the cornea 112 uncross-linked. That is, in theillustrative embodiment, the entire corneal area inside the cornea 112exposed to the cross-linker is selectively cross-linked, thereby leavingthe anterior part of the cornea 112 and the posterior part of the stromauncross-linked. The portion of the cornea 112 without the cross-linkeris not cross-linked when exposed to the UV radiation. In an alternativeembodiment, the cornea 112 may be irradiated using wavelengths of lightother than UV light as an alternative to, or in addition to beingirradiated using the ultraviolet (UV) radiation 122 depicted in FIG.11A. Also, microwave radiation may be used synergistically or additivelyto correct non-invasively the remaining refractive error(s) of thecornea.

Alternatively, as shown in FIG. 11B, a fiber optic 126 may be insertedinto the corneal pocket 116 so as to apply the ultraviolet radiation andactivate the photosensitizer in the wall of the corneal pocket 116. Whenthe fiber optic 126 is used to irradiate the wall of the pocket 116, theultraviolet radiation is applied internally, rather than externally asdepicted in FIG. 11A.

Now, with reference to FIG. 12, it can be seen that, after the wall ofthe corneal pocket 116 has been stiffened and is devoid of cellularelements by the activation of the cross-linkers, a lens implant 128 isinserted into the corneal pocket 116 in order to change the refractiveproperties of the eye. In particular, in the illustrated embodiment, thelens implant 128 is inserted through a small incision, and into thecorneal pocket 116, using forceps or microforceps. In one or moreembodiments, the lens implant 128 that is inserted inside the pocket 116in the cornea 112 is flexible and porous. Also, in one or moreembodiments, the lens implant 128 may comprise a hybrid lens implantwith an organic outer portion and a synthetic inner portion. The organicouter portion of the hybrid lens implant may be made from a transparent,hydrophilic organic polymer, while the synthetic inner portion of thehybrid lens implant may be made from a transparent, gas permeable,porous flexible polymer. For example, the transparent, hydrophilicpolymer forming the organic outer portion may be formed from collagen,chitosan, poloxamer, polyethylene glycol, or a combination thereof (orany other transparent hydrophilic coating which can be deposited overthe entire lens surface), while the flexible polymer forming thesynthetic inner portion of the hybrid lens implant may be formed fromsilicone, acrylic, polymetacrylate, hydrogel, or a combination thereof.The surface of the lens implant 128 may have the appropriate shape toreshape the cornea 112 or the dioptric power to nullify the remainingspheric or astigmatic error of the eye. More particularly, in one ormore embodiments, the lens implant 128 may have one of: (i) a concavesurface to correct myopic refractive errors (i.e., a minus lens forcorrecting nearsightedness), (ii) a convex surface to correct hyperopicrefractive errors (i.e., a plus lens for correcting farsightedness), or(iii) a toric shape to correct astigmatic refractive errors.

In the illustrative embodiment, the irradiation of the cornea 112 usingthe ultraviolet (UV) radiation 122 only activates cross-linkers in theportion of the stromal tissue surrounding the three-dimensional pocket116, and only kills the cells in the portion of the tissue surroundingthe pocket 116, so as to leave only a thin layer of cross-linkedcollagen to prevent an immune response and rejection of the lens implant128 and/or encapsulation by fibrocytes, while preventing post-operativedry eye formation. In addition to preventing encapsulation of the lensimplant 128 by fibrocytes, the cross-linking of the stromal tissuesurrounding the pocket 116 also advantageously prevents corneal hazeformation around the lens implant 128. That is, the cross-linking of thestromal tissue surrounding the lens implant 128 prevents formation ofmyofibroblast from surrounding keratocytes, which then convert graduallyto fibrocytes that appear as a haze, and then white encapsulation insidethe cornea, thereby causing light scattering in front of the patient'seye.

As shown in FIGS. 13 and 14, the crosslinking procedure described abovemay be repeated after the lens implant 128 is implanted so as to preventany cellular invasion in the area surrounding the implant 128.Initially, with reference to FIG. 13, the photosensitizer is reinjectedinside the space between the lens implant 128 and the surroundingcorneal tissue using a needle 120. In one or more embodiments, theneedle 120 for injecting the photosensitizer may comprise a 30-32 gaugeneedle. Then, after the reinjection of the cross-linker, the cornea 112is re-irradiated with ultraviolet radiation 122 to cross-link the tissuesurrounding the lens implant 128 so as to prevent cellular migrationtowards the lens implant 128 (see FIG. 14).

In one or more embodiments, the lens implant or inlay 128 may beprepared ahead of time with known techniques, wherein the inlay 128 maybe coated with a biocompatible material, such as collagen, elastin,polyethylene glycol, biotin, streptavidin, etc., or a combinationthereof. The inlay 128 and the coating may be cross-linked with aphotosensitizer or cross-linker, such as riboflavin, prior to beingimplanted into the pocket 116 in the cornea 112 of the eye.

In another embodiment, the lens implant or inlay 128 may be silicone,methacrylate, hydroxyethylmethacrylate (HEMA), or any otherbiocompatible transparent material, or a mixture thereof. The lensimplant or inlay 128 also may be coated with materials, such as collagenor elastin, and may have a desired thickness of from 2 microns to 70microns or more.

In yet another embodiment, the lens implant or inlay 128 is formed froman eye bank cornea, or a cross-linked eye bank cornea, etc. In general,there is a tremendous paucity of normal cadaver corneas for total orpartial implants, such as for a corneal transplant of a corneal inlay.Because all the cellular elements are killed during the crosslinking ofthe corneal inlay, and because the corneal collagen is cross-linked anddenatured, the remaining collagenous elements are not immunogenic whenimplanted inside the body or in the cornea of a patient. Advantageously,the prior cross-linking of the organic material, such as in the cadavercornea, permits transplantation of the corneal inlay from an animal orhuman cornea or any species of animal to another animal or human for thefirst time without inciting a cellular or humoral response by the body,which rejects the inlay. Thus, cross-linking transparent cadaverictissue for corneal transplantation, or as an inlay to modify of therefractive power of the eye, is highly beneficial to many patients whoare on the waiting list for a corneal surgery. In addition, the surgerymay be planned ahead of time without necessitating the urgency of thesurgery when a fresh cadaver eye becomes available. In one or moreembodiments, the collagens may be driven from the animal cornea, andcross-linked. Also, in one or more embodiments, the implant or inlay 128may be made of cross-linked animal cornea or human cornea that is cutusing a femtosecond laser to any desired shape and size, and thenablated with an excimer laser or cut with a femtosecond laser to a havea desired refractive power.

For example, as shown in FIG. 15, the lens implant or inlay 130 may beformed from an organic block of a polymer (e.g., donor cornea) bycutting the lens implant 130 using an excimer laser (e.g., by using thelaser beam(s) 132 emitted from the excimer laser). Alternatively,referring to FIG. 16, the lens implant or inlay 130′ may be formed froman organic block 134 of a polymer (e.g., donor cornea) by cutting thelens implant 130′ from the block 134 using a femtosecond laser or acomputerized femto-system (e.g., by using the laser beam(s) 136 emittedfrom the femtosecond laser).

In still another embodiment, as depicted in FIG. 17, the lens implant orinlay 130″ is made using three-dimensional (3D) printing technology or amolding technique in order to form the lens implant or inlay 130″ intothe desired shape, size or thickness. The transparent material of the3D-printed implant or inlay 130″ may be coated with one or morebiocompatible polymers and cross-linked prior to the implantation.

In yet another embodiment, after the implantation of an intraocularlens, the remaining refractive error of the eye may be corrected by theimplantation of a lens implant or inlay 128 in the cross-linked pocket116 of the cornea 112, thereby eliminating the need for entering the eyecavity to replace the original intraocular lens.

In still another embodiment, the remaining refractive error of the eyeis corrected after an intraocular lens implantation by placing an inlay128 on the surface of the cornea 112 of the patient while the shape ofthe cornea 112 is corrected with an excimer laser and wavefrontoptimized technology so that the patient is provided instant input onits effect on his or her vision. In this embodiment, an inlay similar toa contact lens is placed on the cornea 112 that, after correction,matches the desired refractive correction of the eye, and then,subsequently, the inlay 128 is implanted inside the cross-linked cornealpocket 116.

In yet another embodiment, the implant or inlay 128 may be ablated withan excimer laser for implantation in the cross-linked pocket 116, orafter cross-linking the exposed corneal stroma in LASIK surgery.

In still another embodiment, a small amount of hyaluronic acid or aviscous fluid is injected into the pocket 116 prior to the implantationof the implant or inlay 128 so as to simplify the insertion of theimplant or inlay 128 in the corneal pocket 116.

In yet another embodiment, the implant or inlay 128 is prepared havingfour marking holes of 0.1-2 millimeter (mm) in diameter in the inlayperiphery at an equally sized distances so that the implant 128 may berotated with a hook, if desired, after the implantation as needed tomatch the axis of an astigmatic error of the eye during the surgery asmeasured simultaneously with a wavefront technology system, such as anOptiwave Refractive Analysis (ORA) system or Holos® system, which arecommercially available for measurement of astigmatism or its axis.

In still another embodiment, the implant or inlay 128 is located on thevisual axis and may provide 1 to 3 times magnification for patientswhose macula is affected by a disease process needing magnifying glassesfor reading, such as in age-related macular degeneration, macular edema,degenerative diseases of the retina, etc. Because these eyes cannot beused normally for reading without external magnifier glasses, providingmagnification by a corneal implant to one eye assists the patients inbeing able to read with one eye and navigate the familiar environmentwith their other eye.

In yet another embodiment, the surface of the cornea 112 is treatedafter surgery in all cases daily with an anti-inflammatory agent, suchas steroids, nonsteriodal anti-inflammatory drugs (NSAIDs),immune-suppressants, such as cyclosporine A or mycophenolic acid,anti-proliferative agents, antimetabolite agents, or anti-inflammatoryagents (e.g., steroids, NSAIDS, or antibiotics etc.) to preventinflammatory processes after the corneal surgery, inlay implantation orcrosslinking, while stabilizing the integrity of the implant 128 andpreventing future cell growth in the organic implant or the adjacentacellular corneal tissue. In this embodiment, the medication is injectedin the corneal pocket 116 along with the implantation or the implant 128is dipped in the medication first, and then implanted in thecross-linked corneal pocket 116.

In still another embodiment, a cross-linked corneal inlay is placed overthe cross-linked corneal stroma after a LASIK incision, and is abated tothe desired size with an excimer laser using a topography guidedablation. By means of this procedure, the refractive power of the eye iscorrected, while simultaneously providing stability to an eye prone toconceal ectasia postoperatively after a LASIK surgery. Then, the LASIKflap is placed back over the implant.

Yet another illustrative embodiment of a corneal lenslet implantationprocedure with a cross-linked cornea is shown in FIGS. 18-23. Ingeneral, the procedure illustrated in these figures involves initiallymaking an intrastromal square pocket surrounding the visual axis of theeye, and then, after forming the initial square pocket, athree-dimensional circular portion of diseased or weak stromal tissue iscut, removed, and replaced with a circular implant which fits into thecircle that borders the four sides of the square. A front view of thecornea 212 of the eye 210 with the centrally-located visual axis 214 isillustrated in FIG. 18. Advantageously, in the illustrative embodimentof FIGS. 18-23, corneal tissue removal around the visual axis is greatlyfacilitated, and nearly perfect centration of the lens implant or inlay220 about the visual axis is possible because the lens implant 220 fitswithin a depressed circular recess at the bottom of the pocket 216. Assuch, the undesirable decentering of the lens implant is prevented.

Initially, in FIG. 19, the forming of an intrastromal square-shapedpocket 216 surrounding the visual axis 214 (represented by a plus sign)in the cornea 212 of the eye 210 is diagrammatically illustrated. Inparticular, as shown in the illustrative embodiment of FIG. 19, thesquare-shaped pocket 216 is formed by making a two-dimensionalintrastromal incision in the cornea 212 of the eye 210 using afemtosecond laser (i.e., the incision is cut in the cornea 212 using thelaser beam(s) emitted from the femtosecond laser).

Then, in FIG. 20, the removal of a three-dimensional circular portion218 of diseased or weak stromal tissue in the cornea 212 of the eye 210is diagrammatically illustrated. In particular, as shown in theillustrative embodiment of FIG. 20, the three-dimensional circularstromal tissue portion 218 has a first diameter, which is less than awidth of the square-shaped pocket 216 so that the three-dimensionalcircular stromal tissue portion 218 is disposed within the boundaries ofthe square-shaped pocket 216. The three-dimensional circular stromaltissue portion 218′ depicted in FIG. 21 is generally similar to thatillustrated in FIG. 20, except that the three-dimensional circularstromal tissue portion 218′ depicted in FIG. 21 has a second diameterthat is slightly larger than the first diameter of the three-dimensionalcircular stromal tissue portion 218 in FIG. 20. As such, the peripheryof the three-dimensional circular stromal tissue portion 218′ depictedin FIG. 21 is disposed closer to the square-shaped pocket 216, but stillwithin the confines of the square-shaped pocket 216. In the illustrativeembodiment, the three-dimensional circular stromal tissue portion 218,218′ may be removed using forceps or micro-forceps. In an exemplaryembodiment, the diameter of the circular stromal tissue portion 218,218′ that is removed from the cornea 212 is between approximately 5millimeters and approximately 8 millimeters, inclusive (or between 5millimeters and 8 millimeters, inclusive).

In an alternative embodiment of the corneal lenslet implantationprocedure, three (3) sequential cuts may be made in the stromal portionof the cornea 212 of the eye 210 using a femtosecond laser in order toform the pocket. First, a lower circular cut or incision centered aboutthe visual axis (i.e., a lower incision with the patient in a supineposition) is made using the femtosecond laser. Then, a second verticalcut is made above the lower incision using the femtosecond laser to formthe side(s) of a circular cutout portion. Finally, a third square orcircular cut (i.e., an upper incision) is made above the vertical cutusing the femtosecond laser. In the illustrative embodiment, the lowerincision is parallel to the upper incision, and the vertical cut extendsbetween lower incision and the upper incision. In this alternativeembodiment, the three-dimensional circular stromal tissue cutout portionbounded by the lower incision on the bottom thereof, the vertical cut onthe side(s) thereof, and the upper incision on the top thereof isremoved from the cornea 212 of the eye 210 using a pair of forceps. Acavity formed by the upper incision facilitates the removal of thethree-dimensional circular stromal tissue cutout portion. As describedabove, the third cut or incision formed using the femtosecond laser maybe an upper circular cut that is larger than the lower circular cut,rather than an upper square cut that is larger than the lower circularcut.

Turning to FIG. 22, after the three-dimensional circular stromal tissueportion 218, 218′ is removed, a photosensitizer is applied inside thepocket 216 so that the photosensitizer permeates the tissue surroundingthe pocket 216. The photosensitizer facilitates the cross-linking of thetissue surrounding the pocket 216. In the illustrative embodiment, thephotosensitizer is injected with a needle 222 inside the stromal pocket216. In one or more embodiments, the photosensitizer or cross-linkerthat is injected through the needle 222 inside the stromal pocket 216comprises riboflavin, and/or a liquid suspension having nanoparticles ofriboflavin disposed therein. Preferably, the cross-linker has betweenabout 0.1% riboflavin to about 100% riboflavin therein (or between 0.1%and 100% riboflavin therein). Also, in one or more embodiments, anexcess portion of the photosensitizer in the pocket 216 may be aspiratedthrough the needle 222 until all, or substantially all, of the excessportion of the photosensitizer is removed from the pocket 216 (i.e., theexcess cross-linker may be aspirated through the same needle 222 so thatthe pocket 216 may be completely emptied or substantially emptied).

Next, turning again to the illustrative embodiment of FIG. 22, shortlyafter the photosensitizer is applied inside the pocket 216, the cornea212 of the eye 210 is irradiated from the outside using ultraviolet (UV)radiation 224 so as to activate cross-linkers in the portion of thetissue surrounding the three-dimensional pocket 216, and thereby stiffenthe cornea 212, prevent corneal ectasia of the cornea 212, and killcells in the portion of the tissue surrounding the pocket 216. In theillustrative embodiment, the ultraviolet light used to irradiate thecornea 212 may have a wavelength between about 370 nanometers and about380 nanometers (or between 370 nanometers and 380 nanometers). Also, inthe illustrative embodiment, only a predetermined anterior stromalportion of the cornea 212 to which the photosensitizer was applied iscross-linked (i.e., the surrounding wall of the corneal pocket 216),thereby leaving an anterior portion of the cornea 212 and a posteriorstromal portion of the cornea 212 uncross-linked. That is, in theillustrative embodiment, the entire corneal area inside the cornea 212exposed to the cross-linker is selectively cross-linked, thereby leavingthe anterior part of the cornea 212 and the posterior part of the stromauncross-linked. The portion of the cornea 212 without the cross-linkeris not cross-linked when exposed to the UV radiation. In an alternativeembodiment, the cornea 212 may be irradiated using wavelengths of lightother than UV light as an alternative to, or in addition to beingirradiated using the ultraviolet (UV) radiation 224 depicted in FIG. 22.Also, microwave radiation may be used synergistically or additively tocorrect non-invasively the remaining refractive error(s) of the cornea.In addition, in an alternative embodiment, the ultraviolet (UV)radiation may be applied after the implantation of the lens implant 220to perform the crosslinking, rather than before the implantation of thelens implant 220 as described above. Further, rather than applying theultraviolet (UV) radiation from outside the cornea 212, the stromaltissue of the pocket 216 may be irradiated from inside by means of afiber optic, before or after the implantation of the lens implant 220.

Now, with combined reference to FIGS. 22 and 23, it can be seen that,before or after the wall of the corneal pocket 216 has been stiffenedand is devoid of cellular elements by the activation of thecross-linkers, a circular lens implant 220 is inserted into the circularrecess at the bottom of the pocket 216 formed by the three-dimensionalcircular stromal tissue cutout portion 218, 218′ that was removed. Thatis, the circular lens implant 220 fits within the periphery of thecircular recess that borders the four sides of the squared-shaped pocket216. In particular, in the illustrated embodiment, the circular lensimplant 220 is inserted through a small incision, and into the circularrecess at the bottom of the pocket 216 using forceps or microforceps. Inthe illustrative embodiment, the flexible lens implant 220 may befolded, inserted through the small incision, placed inside the circularrecess at the bottom of the pocket 216, and finally unfolded throughthen small incision. In one or more embodiments, the lens implant 220that is inserted inside the pocket 216 in the cornea 212 is flexible andporous. Also, in one or more embodiments, the lens implant 220 maycomprise a hybrid lens implant with an organic outer portion and asynthetic inner portion. The organic outer portion of the hybrid lensimplant may be made from a transparent, hydrophilic organic polymer,while the synthetic inner portion of the hybrid lens implant may be madefrom a transparent, gas permeable, porous flexible polymer. For example,the transparent, hydrophilic polymer forming the organic outer portionmay be formed from collagen, chitosan, poloxamer, polyethylene glycol,or a combination thereof (or any other transparent hydrophilic coatingwhich can be deposited over the entire lens surface), while the flexiblepolymer forming the synthetic inner portion of the hybrid lens implantmay be formed from silicone, acrylic, polymetacrylate, hydrogel, or acombination thereof.

Advantageously, the lens implant 220 of the aforedescribed illustrativeembodiment always remains perfectly centered around the visual axis 214of the eye 210, and will not move because it is disposed within thecircular recess at the bottom of the pocket 216. As explained above, thelens implant 220 may be formed from an organic material, syntheticmaterial, polymeric material, and combinations thereof. The lens implant220 may replace either a diseased tissue or create a new refractivepower for the eye 210, as explained hereinafter.

In the illustrative embodiment, the lens implant 220 may correct therefractive errors of the eye 210. The refractive error correction may bedone by the lens implant 220 having a curvature that changes the cornealsurface of the cornea 212. Alternatively, the lens implant 220 may havea different index of refraction that corrects the refractive power ofthe cornea 212. In the illustrative embodiment, the lens implant 220 mayhave the appropriate shape to reshape the cornea 212 or the dioptricpower to nullify the remaining spheric or astigmatic error of the eye.More particularly, in one or more embodiments, the lens implant 220 mayhave one of: (i) a concave anterior surface to correct myopic refractiveerrors (i.e., a minus lens for correcting nearsightedness), (ii) aconvex anterior surface to correct hyperopic refractive errors (i.e., aplus lens for correcting farsightedness), or (iii) a toric shape tocorrect astigmatic refractive errors.

In the illustrative embodiment, the irradiation of the cornea 212 usingthe ultraviolet (UV) radiation 224 only activates cross-linkers in theportion of the stromal tissue surrounding the three-dimensional pocket216, and only kills the cells in the portion of the tissue surroundingthe pocket 216, so as to leave only a thin layer of cross-linkedcollagen to prevent an immune response and rejection of the lens implant220 and/or encapsulation by fibrocytes, while preventing post-operativedry eye formation. In addition to preventing encapsulation of the lensimplant 220 by fibrocytes, the cross-linking of the stromal tissuesurrounding the pocket 216 also advantageously prevents corneal hazeformation around the lens implant 220. That is, the cross-linking of thestromal tissue surrounding the lens implant 220 prevents formation ofmyofibroblast from surrounding keratocytes, which then convert graduallyto fibrocytes that appear as a haze, and then white encapsulation insidethe cornea, thereby causing light scattering in front of the patient'seye.

In the further illustrative embodiments described hereinafter, thecornea is cross-linked soon after the initial corneal transplant where asuture is placed around the implant to keep the implant in place untilthe tissue heals between the host and the corneal transplant, before thegraft and often the suture itself can act as a foreign body inducing animmune response with neovascularization of the corneal transplant whichwill be rejected and become scarred. For example, in the aforedescribedembodiments, the cross-linking may be performed between two and threemonths after transplantation of the corneal graft or prosthesis in orderto allow the transplantation area time to heal before the cross-linkingprocedure is performed.

A fifth illustrative embodiment is depicted in FIGS. 24 and 25. In thisillustrative embodiment, shortly after corneal transplantation (see FIG.24) the remaining peripheral host cornea 302 of the eye 300, the suture304 and the transplant (i.e., the corneal graft 306) is treated withdrops of 0.05-1% or more of riboflavin, CPP/nanoparticles, physiologicalsolution or the use of another photosensitizer or cross-linking agent,that is applied to the cornea 302 for a period of a few minutes to 30minutes. Then, the entire cornea 302 is irradiated with a UV laser 310emitting ultraviolet radiation 308 of 370-400 nm wavelength, 1-30 mw/cm2for a desired time of 1-30 minutes, depending on the concentration ofthe photosensitizer and the power of the laser light to cross-link thecorneal collagen and damage the keratocytes in it, in at least one-thirdof the front thickness of the host cornea and the transplant to preventan immune response to the transplant and its rejection (refer to FIG.25).

A sixth illustrative embodiment is depicted in FIGS. 26 and 27. In thisillustrative embodiment, the neovascularization 406 has started or hasreached the edges of the transplanted cornea 404 with sutures 412 (seeFIG. 26), one applies riboflavin or another photosensitizer to theanterior part of the cornea 402 of the eye 400 prior to the cross-linkermolecule diffusing to the posterior corneal layers and the cornealendothelial cells under observation with a slit lamp, then the cornea402 is irradiated with UV light 408 from a UV laser 410 as describedpreviously to cross-link at least one half of the anterior thickness ofthe cornea 402.

A seventh illustrative embodiment is depicted in FIG. 28. In thisillustrative embodiment, the repeated corneal transplant has led to acloudy vascularized cornea 502 in which no corneal transplantation canbe considered, the cornea 502 of the eye 500 is prepared forimplantation of a keratoprosthesis lens 504 by removing the center partof the opaque cornea 502 by a trephine, thus creating a central openingin the cornea 502, and then a circular pocket in the remainingperipheral cornea is produced horizontally with a knife or laser so asto produce an anterior and a posterior flap around the central openingin the remaining part of the peripheral cornea, in which a prostheticlens 504 with a central portion 504a and a peripheral flange 504b isimplanted. As shown in FIG. 28, the peripheral flange 504b of theprosthetic lens 504 is provided with a plurality of apertures or holes506 disposed therethrough for allowing the aqueous humour fluids of theeye to provide nutrients to the donor graft stroma. The riboflavin dropsor nanoparticle suspension of riboflavin with or without CPP are appliedto penetrate the front of the cornea 502 and the posterior corneal flap,followed a few anchoring 8-0 or 10-0 nylon sutures 512 to hold theprosthetic lens 504 in place, then followed by UV radiation 508 from aUV laser 510 of the entire cornea 502, except the lens 504 which iscovered with a piece of tissue paper to prevent the light from gettinginside the eye 500, thereby preventing vascular growth in the prosthesis504 from the front and its side or fibrous tissue growth to the backside of the prosthesis 504 that could block the light reaching theretina (refer to FIG. 28).

Next, illustrative corneal crosslinking procedures with enhancedpenetration of the crosslinking agent will be described. The process ofcrosslinking can be very time consuming in which the penetration of thecross-linker (e.g., riboflavin or any other cross-linker) is applied asdrops to the corneal tissue for about 15-30 minutes to penetratetypically a depth of 150-200 microns of the anterior corneal stroma.Considering that corneal crosslinking often requires UV radiation for aperiod of 30 minutes, the operation takes at least between 30-60 minutesto perform. Also, riboflavin does not penetrate the cell membrane well.In the above-described embodiments, because the purpose is to reduce andeliminate the potential of corneal transplant rejection by the survivedhost corneal epithelial cells or the host stromal cells that producecytokines against the transplanted cornea and vice versa, it isdesirable to eliminate the cellular component of the host cornea or thetransplanted cornea, or both, while maintaining the host cornealendothelial cells intact. Since the corneal endothelial cells, ingeneral, are not affected if the riboflavin is applied over the cornealsurface through which it penetrates inside the stroma, it is desirableto enhance cellular penetration of the cross-linker, and simultaneouslypenetration through the corneal stroma, which is made of collagen.Although the procedures described above can be used with standardriboflavin or any other crosslinking solution, a technique andformulation for expediting the cell penetration of riboflavin and thecrosslinking of the cornea for corneal crosslinking and elsewhere in thebody is very desirable.

In one or more embodiments, cell penetrating peptides are used thatcomprise the short peptide lysine or arginine, which are known as cellpenetrating peptides (CPP) and activatable-cell penetrating peptides(ACPP). CPP and ACPP may be conjugated to dendrimers (ACPPDs) or othernanoparticles (e.g., riboflavin) or any other cross-linkers. The ACPPmay be labeled with a polycationic CPP. ACPP and CPP may benaturally-occurring or artificially constructed protein segments (<30amino acids) rich in arginine, lysine, cysteine, histidine, ornithine,etc.; preferably .alpha.-helices and about 17-amino acids. The ACPP andCPP may include a penetration accelerating peptide sequence (Pas) or anINF7 fusion peptide sequence. CPP and/or ACCP can be linked to cargoeseither covalently or non-covalently. Nanoparticles may be delivered bycell-penetrating peptides comprised of nona-arginine and a penetrationaccelerating sequence. Also, nona-arginine may be used to facilitate thedelivery of the riboflavin cross-linker into cells via multiplepathways. Exemplary, but not limiting ACPP and CPP may includetransportan, penetratin, TAT, VP22, MAP, KALA, ppTG20, proline-richpeptides, MPG-derived peptides, Pep-1, nona-arginine, and thecarboxy-terminal tail of TFPI-2, polyproline helices having cationicamino acids and/or cationic-functionalized amino acids within thehelix). Nanoparticles may be coated or otherwise associated with organicor non-organic biodegradable compounds, aliphatic biodegradablepolymers, as needed. The nanoparticles may comprise organicnanoparticles, non-organic nanoparticles, synthetic nanoparticles, ornon-synthetic nanoparticles.

In one embodiment, riboflavin or other cross-linkers may be linked to,associated with, complexed or conjugated with nanoparticles usinglinking agents and methods including but not limited to the following:amino groups, carboxyl groups, S—S deprotected sulfhydril groups inbiomolecules, carbodiimide conjugation, sulfosuccinimidylsuberyllinkage, synthetic tripyrrole-peptide linkage, NHS-esters and otheresters, etc.

In one embodiment, the riboflavin or a cross-linker conjugated todendrimers (ACPPDs) is applied to a cornea which has developedneovascular tissue, where the penetration of the riboflavin is veryminimal in the endothelial cells or the neovascular tissue, so thatsubsequent UV radiation will not damage these cells or close theneovascular tissue of the cornea. Dendrimers and other types ofnanoparticles do not need to have an antibody attached to them as longas they are applied topically for the crosslinking of the cornea.Although, nanoparticles do need to have a specific antibody attached tothem if they are injected in the circulation of the patient fortargeting a tumor.

In one embodiment, the cross-linker is conjugated with nanoparticles inthe form of dendrimers or functionalized dendrimers conjugated with CPPor ACPP and administered locally, topically or injected in body cavity,to be absorbed by normal or abnormal tissue or tumor with theirneovascular tissue and subsequently cross-linked by UV radiation orother laser wavelength absorbed by the cross-linker, not only damagingthe neovascular tissue, but also the tumor cells, as an example. Also,the cross-linker conjugated with the dendrimers or functionalizeddendrimers conjugated with CPP or ACPP may be applied to a surfacelesion/tumor on the skin, mucosa or conjunctiva of the eye or inside abody cavity.

In one embodiment, the nanoparticles carrying riboflavin or anothercross-linker are made of lactic acid, glycolic acid, or polycaprolactoneand conjugated with a cell specific or organism specific antibody toattach to their cell membrane receptors.

In one embodiment, the nanoparticles are made of lactic acid, glycolicacid, polycaprolactone, or chitosan, or are in the form of dendrimers,and are conjugated with riboflavin, CPP and antibodies to targetspecific cells, such as tumors of neovascular cells, and to attach tothe cell membrane receptors of these cells or to one or more organismsand to penetrate the cell walls of specific cells or organisms, thuscarrying the photosensitizer or riboflavin inside the cell, whileactivated with a light or UV light, thereby crosslinking the cytoplasmicproteins and kill the cells or the organism.

In one embodiment, the photosensitizer is in the form of drops, and thedrop solution or suspension of nanoparticles or dendrimers comprisesbetween about 0.05% and about 1% riboflavin or a photosensitizertherein.

In one embodiment, the photosensitizer or riboflavin is in the form ofdrops or suspension of nanoparticles, and the drop solution comprisesbetween about 0.0005% and about 5% riboflavin therein.

In one embodiment, riboflavin or the cross-linker solution may haveethylenediaminetetraacetic acid (EDTA) or Disodium Edetate Dihydrate 0.1w/w %, Sodium Chloride 0.4 w/w %, Polycarbophil 0.95 w/w %, Octoxynol 40(70% Solution), 2N NaOH (active dissolution @pH 7.9), q.s. to adjust pHto about 7.4-7.7 as desired, Mannitol 0.15 w/w %, Sodium thiosulfate 0.3w/w %, Water q.s. to 100%. The riboflavin or the cross-linker solutionmay have an osmolarity of about 300 mOsm/L. The riboflavin or thecross-linker solution may also contain a cell penetrating agent (e.g.,CPPs or ACPPs). In addition, the riboflavin or the cross-linker solutionmay be provided with or without a poloxamer and with or without dextran.

In one embodiment, the cross-linker is applied through a circular gelwith the desired diameter of 1 millimeters (mm) to 15 millimeters (mm)or more to be placed on the surface of the cornea or other surfaces sothat the cross-linker penetrates the desired area of the corneaselectively, etc.

In one embodiment, the gel has a thickness of 0.01 mm to 3 mm or more.

In one embodiment, the diameter of the gel is 5-10 mm.

In one embodiment, the gel is circular with an opening of 1-8 mm.

In one embodiment, the gel is made of an organic or synthetic material.

In one embodiment, the gel is made from cellulose derivatives.

In one embodiment, the gel is soaked with the photosensitizer, such asriboflavin or another chemical, at a desired concentration that candiffuse from it inside the tissue within the desired time.

In one embodiment, the circular gel fits on the surface of the cornea oranother desired surface and has strategically made holes to selectivelyrelease the photosensitizer in that area, so that during thecrosslinking, only specific areas are selectively cross-linked, whilethe other areas are left alone to have specific effect either in therefractive power, elasticity on the cornea or for specific selectiveimplantation of an implant.

In one embodiment, the cross-linker or riboflavin/CPP are conjugatedwith functionalized nanoparticles, such as acrylic or acrylicderivatives or crystalline silicon or other organic transparentnanoparticles, to enhance cell penetration and penetration of thenanoparticles through the corneal stroma to kill the stromal cells andentrap the acrylic or crystalline silicon nanoparticle in thecross-linked stromal collagen and inside the cells after irradiationwith UV radiation applied externally of through a fiber optic andcrosslinking their cytoplasmic proteins and the collagen. Additionally,the corneal stroma and nanoparticles's index of refraction can bechanged in the same session or later, using a femtosecond ormulti-photon laser and a Shack-Hartmann unit to achieve a perfectrefractive power for the cornea.

In one embodiment, a corneal inlay is soaked in a solution having across-linker or riboflavin/CPP are conjugated with coated nanoparticles,such as acrylic or acrylic derivatives or crystalline siliconnanostructure or organic transparent nanoparticles, to enhance cellpenetration and penetration of the nanoparticles through the cornealstroma to kill the stromal cells and entrap the acrylic or crystallinesilicon nanoparticle in the cross-linked stromal collagen and inside thecells after irradiation with UV radiation applied externally beforeimplanting inside a corneal pocket or after implantation inside thestromal pocket using a fiber optic and crosslinking their cytoplasmicproteins where the corneal stroma and subsequently the refractive indexof the inlay and the cornea with the nanoparticles can be changed usinga femtosecond or multi-photon laser and a Shack-Hartmann unit to achievea perfect refractive power for the cornea or modifying it forastigmatic, presbyopic, myopia, or hyperopia correction.

In one embodiment, the corneal inlay is prepared from a 3-D stromaltissue culture or an eye bank eye cornea where the inlay is soaked in asolution having a cross-linker or riboflavin/CPP are conjugated withPEGylated nanoparticles, such as acrylic or acrylic derivatives orcrystalline silicon nanostructure or organic transparent, organicnanoparticles to enhance cell penetration and penetration of thenanoparticles through the corneal stroma to kill the stromal cells andentrap the acrylic nanoparticle or crystalline silicon in thecross-linked stromal collagen and inside the cells after irradiationwith UV radiation applied externally before implanting inside a cornealpocket or after implantation inside the stromal pocket using a fiberoptic and crosslinking their cytoplasmic proteins where the cornealstroma and subsequently the refractive index of the inlay or the corneawith the nanoparticles can be changed using a femtosecond ormulti-photon laser and a Shack-Hartmann unit to achieve a perfectrefractive power for the cornea.

In one embodiment, the cross-linker or riboflavin/CPP are conjugatedwith antibody coated nanoparticles or dendrimers, to reach specificcells targeted to kill after irradiation with UV radiation appliedexternally of through a fiber optic and crosslinking their cytoplasmicproteins, etc.

In one embodiment, the targeted cells are tumor cells.

In one embodiment, the tumor cells are located on the skin or mucosa.

In one embodiment, the riboflavin/CPP conjugated with antibody coatednanoparticles are injected inside the body cavity where thenanoparticles are attached to the tumor cells, then treated with UVradiation brought in using a fiber optic crosslinking their cytoplasmicproteins, etc.

In one embodiment, the riboflavin/CPP conjugated with antibody coatednanoparticles are administered intravenously to reach an internallylocated tumor and to attach to their cell membranes and kill them afterlocally administered UV radiation.

In one embodiment, the tumor is in the mouth, nose, throat, eye,conjunctiva, or lid of the eye, or lung and can be reached with a UVlaser with a fine fiber optic to irradiate the tumor and kill them bycrosslinking their cytoplasmic proteins, etc.

In one embodiment, the lesion is an infected ulcer of the cornea, skinor mucosa, nasal, throat, etc. that can be treated by topicalapplication of the riboflavin/CPP or ACPP conjugated with antibodycoated nanoparticles to kill the bacteria, viruses, fungi, protozoalinfections treated with UV radiation applied through a fiber optic andcrosslinking their cytoplasmic proteins, etc.

In one embodiment, the lesion is an infected ulcer of the cornea, skinor mucosa, nasal, throat, etc. that can be treated by topicalapplication of the riboflavin/CPP or ACPP conjugated with antibodycoated nanoparticles or dendrimers to kill the bacteria, viruses, fungi,protozoal infections treated with UV radiation applied through a fiberoptic and crosslinking their cytoplasmic proteins, etc.

In one embodiment, the ulcer is an infected corneal ulcer treated bytopical application of the riboflavin/CPP or ACPP conjugated withantibody coated nanoparticles or dendrimers to kill the bacteria with UVradiation applied through a fiber optic or an external UV light andcrosslinking their cytoplasmic proteins, etc. as described before.

In one embodiment, the ulcer is an infected skin or mucosal ulcertreated by topical application of the riboflavin/CPP or ACPP conjugatedwith PEGylated nanoparticles/dendrimers to kill the bacteria, with UVradiation applied through a fiber optic or an external UV light bycrosslinking their cytoplasmic proteins, etc., as described before.

In one embodiment, the keratoprosthesis 504 described above is coatedwith biocompatible nanoparticles, such as gold or silica, or acombination thereof, etc. in a manner that does not cover the centraloptical lens to prevent rejection.

In one embodiment, the riboflavin nanoparticle/dendrimer and CPP is usedas a surface coating for any intracorneal implantation followed with UVradiation to kill the cells surrounding it by crosslinking theircytoplasmic proteins etc.

In one embodiment, the riboflavin nanoparticle or dendrimers and CPP isadministered after extracapsular lens removal followed by administrationof CPP/cross-linker conjugated with polyethylene glycol (PEG) coatednanoparticles in the capsular bag, then followed by lens implantationwhere the CPP enhances the penetration of the riboflavin into the lensepithelial cells and kills them by crosslinking their cytoplasmicproteins etc., thereby preventing capsular opacification.

In one embodiment, a glaucoma stent surface is coated with biocompatiblenanoparticles, such as gold or silica, or a combination thereof, etc. toprevent rejection of the stent, then coated with CPP/riboflavinnanoparticles or dendrimers prior to the implantation of the glaucomastent and then irradiated with UV radiation after the implantation tokill the cells by crosslinking their cytoplasmic proteins, etc. aroundthe stent and prevent ingrowth of the cells blocking the stent andprevent ingrowth of the cells blocking the stent using a fiber optic.

In one embodiment, a cardiac/vascular stent is coated with biocompatiblenanoparticles, such as gold or silica, or a combination thereof, etc. toprevent rejection, then coated with CPP/riboflavin nanoparticles ordendrimers prior to the implantation of the vascular stent andirradiated with UV radiation after the implantation to kill the cells bycrosslinking their cytoplasmic proteins, etc. around the stent andpreventing ingrowth of the cells blocking the stent using a fiber optic.

It is readily apparent that the aforedescribed corneal transplantprocedures and inlay implantation procedures offer numerous advantages.First, the implementation of the aforedescribed corneal transplantprocedures reduces the likelihood that the implanted cornea will berejected by the patient. Secondly, the aforedescribed corneal transplantprocedures enable the clarity of the transplanted cornea to bepreserved. Finally, the aforedescribed corneal transplant proceduresreduce the likelihood that the transplanted cornea will be invaded bymigrating cells, such as migrating cells that might initiate an immuneresponse such as macrophage, lymphocytes or leucocytes or vascularendothelial cells. These types of migrating cells are discouraged by thecross-linked corneal collagen which does not provide an easilyaccessible tissue to invade. In addition, the use of abovedescribedtissue adhesives reduces the surgical procedure significantly. Moreover,the aforedescribed corneal lenslet implantation procedures modify thecornea so as to better correct ametropic conditions. Furthermore, thecorneal lenslet implantation procedures described above prevent the lensimplant from moving around inside the cornea once implanted, therebyensuring that the lens implant remains centered about the visual axis ofthe eye. In addition, the aforedescribed inlay implantation proceduresprevent an immune response to the corneal inlay and to prevent arejection of the corneal inlay by the patient.

Illustrative embodiments of a drug delivery implant and methods usingthe same will now be described hereinafter. In accordance with thevarious embodiments described herein, in order to provide the medicationto the anterior and posterior part of the eye with a slow release drugsystem, it is required to create an immune privileged space inside thecornea to keep the cellular response away and prevent production ofcytokine by them, and position the device outside the central visualaxis so that the device would not interfere with the patient's vision.

In the embodiments described herein, the device is placed in the farcorneal periphery so that it will not affect the vision or visual fieldof the patient, and so that it has created a so-called artificial“immune-privilege” which does not generate an immune response from thebody while fluid, soluble medications or nano-particulates andmicro-particulates can travel through it. See, for example, FIGS.38A-45B.

Because of the location of the implant inside the cornea, the releasedmedication bypasses the epithelial barrier of the cornea, whileproviding medication in a slow manner by diffusion to the anterior partof the cornea, to the sclera, to the conjunctival tissue, and to theposterior segment of the eye including the retina, choroid, and theoptic nerve head. This technique can provide similar immune-privilegedspaces in other part of the body so that devices implanted there are notencapsulated.

The drug delivery system of the embodiments described herein may beconstructed so that it can have direct access to the anterior chamber,if needed, for both obtaining repeatedly a fluid biopsy from the eye ordeliver medication(s) directly inside the eye in a fast or slow releasemanner, or for reducing the intraocular pressure of the eye by creatinga minor flow through a porous implanted stent or tube through thecorneal limbus without inducing a fibrous encapsulation of the stent.The stent can ameliorate also corneal dryness caused by dry eyesyndrome. The stent can also be equipped with a pressure sensorindicating directly the intraocular pressure and communicating it with aradiofrequency device to outside the eye to a receiver or a processor.As one example, as shown in FIG. 37, the implant 644 may comprise aclosed end 646 and a needle 648 for tissue penetration so that theimplant 644 is capable of being used for taking liquid biopsies. Inaddition, stem cells or other cells can reside in, for example, atubular implant, while having access to the oxygen and nutrients throughthe artificial barrier in an appropriately prepared corneal pocket.However the porous tubular implant permits these cells to migrateelsewhere in the eye or remain in place without being attacked by body'scellular response. Because the cellular body immune response isdependent on the production of the cells close to the implant or aforeign body to be taken up by the dendritic cells of the body at thatlocation by creating a cell free space around the implant made oftransparent amorphous cross-linked collagen. The invention of theembodiments described herein has eliminated the incentive for a MajorHistocompatibility Complex (MHC) to occur. Because these MHC are presenton the cell surface of the body cells to be activated in the productionof an immune response. The release of theses cytokines activates thecellular immune system of the body to either eliminate the threat orisolate the device from the body completely by fibrocytes, therebybuilding a dense membrane (i.e., scar) around the implant. However, thecross-linked collagen permits the diffusion of water and small moleculespermitting the needed growth factors from the incorporated stem cellsplaced inside the tubular implant needed for survival and the health ofthe cornea, retina etc.

Though this mechanism is very effective and useful, it affects thefunction of an implant that usually either releases a needed medicationor measures or controls the release of a medication (e.g., measuring theblood glucose level and/or releasing insulin according to the glucoselevel found in the blood, etc.).

In order to isolate an implant in the body while preventing the immunecell to gain access to the device or build a membranous scar tissuearound it, a method has been developed to isolate the implant in thebody by killing all the cells adjacent to an implant, while maintaininga fluid-filled area around the implant or creating a barrier out of thesurrounding tissue containing collagen and cross-linking the tissue invivo. This barrier protects the implant from the antigen presentingdendritic cells in the tissue, while permitting the soluble medicationor nano-sized particulate material to pass through the barrier so as totreat a pathological process in the body. One can also monitor the levelof the analytes in the tissue fluid (e.g., aqueous fluid levels ofglucose), which is a representative of the blood glucose level in theblood, from which it is originated. Aqueous level of most if notanalytes found in the blood and could be used effectively to provideinformation on the health or disease processes affecting the eye or thebody as a whole.

In the embodiments herein, implantation of a drug delivery device isdescribed for the release or monitoring and controlling of a diseaseprocess in the eye, while crosslinking the tissue around the implant orimplants (if more than one implant is provided). In any of theembodiments described herein, a plurality of drug delivery implants maybe used (e.g., for delivering different medications), rather than asingle drug delivery implant.

The technology described herein may be applied for any other deviceimplantation in the body regardless of the location in the body. One ofthe benefits of the technology is that, if the device needs to bereplaced, it can be done easily without dealing with the scar tissueformation that otherwise forms and makes the removal or replacement ofthe implant very complex because the tissue adhesions that usually formsbetween the tissue and the device.

One can use this concept described herein for diagnosis or therapy indiseases affecting the cornea, a metabolic disorder, genetic disorder,glaucoma, an infection affecting the eye or another portion of the body,a disease or disorder affecting the front or the back part of the eye orthe conjunctiva or lens, an aging process, such as dry eye formation,retinal diseases including infective processes, genetic diseasesrequiring gene therapy (e.g., retinitis pigmentosa, etc. or metabolicdisorders such as diabetes, etc.).

In one embodiment, if the media is clear, a two dimensional intrastromalcorneal incision is created that is subsequently converted into a pocketin the corneal stroma using a femtosecond laser or a mechanical cuttingsystem. The femtosecond laser passes through the clear media of thecornea. When the laser beam is focused inside the cornea, one canproduce a two-dimensional cut or a three-dimensional cut around a thinpart of the tissue that is removed to desired space, shape, depth, andlocation.

In another embodiment, in opaque elastic tissue (e.g., skin), one canuse a knife or a syringe needle ending in a sharp cutting tip to cut apocket in the tissue. If needed, the incision simultaneously involvesremoval of a three-dimensional tissue surrounding the surgical pocket tocreate some additional space for the implant using a similar cuttinginstrument, in the skin or soft tissue. In general, a cut creates aflexible three-dimensional space that can be filled with an implant. Theimplant is placed inside the needle and can be expelled from the needleby the syringe into the space created by knife.

In one or more embodiments, an injectable anesthetic (e.g., lidocaine orBupivacaine) in a desired non-toxic preparation or concentration of0.1-2% or more in a physiologic solution with, but preferably without, apreservatives, is injected in the corneal pocket to anesthetize thecornea postoperatively for a period up to 8-12 hours (e.g., if a PRKprocedure is contemplated or after a corneal inlay implantation toprevent pain sensation completely in the postoperative period). Thiseliminates subjecting the entire corneal epithelium or the conjunctivalepithelial cells to the damaging effect of topical anesthesia, whichdelays corneal re-epithelialization or conjunctival epithelial cells.Generally, the topical preservatives present in the topical anesthesiadamages the cells that are bathed in them, and at times affects theregeneration of these cells (i.e., corneal epithelial or conjunctivalcells) if applied frequently. Also, it may produce addiction to thetopical anesthesia for eliminating the pain sensation caused by the lossof the corneal epithelial cells, whereas the injectable anesthetic doesnot damage the epithelial cells, including the nerve cells or theiraxons, except for blocking temporarily the neuronal transmission.

In one or more embodiments, the collagen cross-linker is mixed with theintracorneal locally injectable anesthetic, and injected simultaneouslyor sequentially in the corneal pocket.

In one or more embodiments, the pocket is filled with a biocompatibleimplant or implants (if more than one implant is provided) made oforganic or non-organic material, or a mixture of it, and the implant isused for drug delivery. The implant may further be coated with abiocompatible material, such as collagen, elastin, polyethylene glycol,biotin and streptavidin, etc., as known in the art, or a compositionthereof, to make the implant more biocompatible. The implant and/or thecoating can be cross-linked with a cross-linker with the desiredthickness and shape before or after implantation.

In one or more embodiments, the diameter of the corneal pocket can be0.1 to 4 millimeters (mm), as needed. Only flat implants need a largerspace with more than 0.2 mm. As shown in FIG. 38A-45B, the pocket can becircular, semicircular, C-shaped, doughnut-shaped, rectangular, or anyother shape.

In one or more embodiments, the implant or implants (if more than oneimplant is provided) can be located at a desired distance from theBowman's membrane or from the corneal periphery, that is located awayfrom the center of the visual axis (i.e., the implant may beoff-centered, or ring-shaped in the peripheral cornea). See, forexample, FIGS. 38A-45B.

In one or more embodiments, the implant or implants (if more than oneimplant is provided) is made to the desired shape, and size in diameterand length that fits with ease inside the corneal pocket withoutexerting pressure on the corneal tissue (i.e. without bulging it).

In one or more embodiments, a photosensitizer or cross-linker, such asriboflavin, is injected at the desired concentration in a biocompatiblefluid or a viscous fluid prior to the implantation of the implant.However, it can be also administered simultaneously with the implant inthe corneal pocket sufficiently to cover the internal wall of the pocketfor a desired duration so that it penetrates at least 20 micron orwider, taking 5-30 seconds after injection prior to the cross-linking ofthe cornea, which prevents cell proliferation, encapsulation, orrejection of the implant while preserving an acellular barrier.

In one or more embodiments, ultraviolet (UV) radiation at the desiredpower (e.g., 1 to 4 mW/mm²) and duration of 1-15 minutes, as needed,depending on the concentration of the photosensitizer or other radiationif another cross-linker is used (e.g., visible or infrared (IR) oranother wave length) is applied externally to activate thephotosensitizer in the corneal pocket, and to cross-link the collagen ofthe corneal stroma surrounding the corneal pocket, thereby killing onlythe cells located within the cross-linked cornea while preventingencapsulation of the drug implant while providing a physical stabilityto the cornea and preventing the wall of the pocket from adheringtogether or to the implant. This permits the implant to be replaced, ifneeded, with another implant with ease.

In one or more embodiments, the implant is coated with an organicmaterial, such as collagen, dipped in a photosensitizer, or the implantcan be coated with nanoparticles of the photosensitizer and implanted inthe corneal pocket and ultraviolet (UV) radiation is applied with thedesired power and duration using a painting technique using a smalldiameter fiber optic or other radiation with another wave length isapplied if another cross-linker is used, externally or internally insidethe pocket via a fiber optic to activate the photosensitizer in thecorneal pocket and to cross-link the collagen of the corneal stromasurrounding the corneal pocket, thereby killing all cells located withinthe cross-linked cornea and cross-link the implant simultaneously. Thecorneal cross-linking prevents implant encapsulation with fibroustissue, but provides a physical stability to the cornea without gluingthe wall of the pocket together or to the implant.

In one or more embodiments, an injection of a small amount of hyaluronicacid in the pocket simplifies insertion of the drug implant in thecorneal pocket.

In one or more embodiments, the drug implant has a tube-like structurewith a size of 0.01 to 3 micron diameter holes in its wall, or havingone micron or larger-sized holes for diffusion of fluid across it.

In one or more embodiments, the implant can be silicone, acrylic,methacrylate, hydroxyethyl methacrylate (HEMA), cross-linked organic orany other biocompatible transparent or non-transparent material,metallic or non-metallic, or a mixture thereof or coating otherpolymers, such as collagen or elastin with the desired thickness of 2microns or more, as needed.

In one or more embodiments, the implant is made of various drug deliverypolymers, such as polylactic acid or polyglycolic acid, or a combinationthereof or polycaprolactone, or chitosan or other organic materials thatcan deliver the medication at a certain concentrations and dissolvewithin time ranging from 3-12 months or more.

In one or more embodiments, the biodegradable or non-biodegradableimplant can be replaced with another one as before or anon-biodegradable material, but having biocompatible material or coatingwhere the drug release occurs either through the small holes in the bodyof the implant at a certain rates depending on the size of the holes, orfrom one or both ends of the implant for drug delivery, as needed.

In one or more embodiments, the implant is a porous biodegradablepolymer.

In one or more embodiments, the material inside the tubular implant isliquid, nanoparticles, suspension, powder, porous polymeric drug, etc.

In one or more embodiments, the implant is made using 3-D printingtechnology to the desired shape, size and/or coated with morebiocompatible polymer(s) and cross-linked prior to the implantation, orit is implanted in a cross-linked pocket.

In one or more embodiments, the cross-linked corneal implant can beloaded with one or multiple medications needed for a short biocompatibledrug delivery, or prophylactically to prevent an infection, or otherused therapeutically medications to treat a disease process (e.g.,inflammation, intraocular pressure (IOP), neovascularization, infection,or a cytokine, etc.).

In one or more embodiments, an organic cross-linked material can be usedas above for a short term drug delivery of 1 to 4 weeks.

In one or more embodiments, an organic cross-linked material can be usedas above for a short term drug delivery of 5 to 50 weeks or longer.

In one or more embodiments, the implant is a C-shaped flexible orsemi-flexible structure, and can be implanted in the prepared cornealpocket according to the size or the shape of the implant (e.g., centeredaround the visual axis having a string shape, rod-like shape, or flatshape), while removing a small 3-D tissue from the stroma for the pocketformation to provide space for the implant for drug delivery to thecornea or the anterior chamber, trabecular meshwork, conjunctiva, ordiffusing toward the posterior segment, such as the retina, choroid orthe optic nerve of the eye. As shown in FIGS. 29A-29D, the drug deliveryimplant may be rod-shaped 610, C-shaped 612, two-part semi-circular 614,or one-part semi-circular 616. Also, as illustrated in FIG. 34, theimplant may also be in the form of a rectangular flat tube 634. In FIGS.38A and 38B, a two-part semi-circular drug delivery implant 656 disposedin a cross-linked pocket in the peripheral portion of the cornea 652that is spaced apart from the central visual axis 654 of the eye so asnot to obstruct the central portion of the eye. As shown in FIG. 38B,the two-part semi-circular drug delivery implant 656 is disposedadjacent to the anterior chamber 657 of the eye, and anteriorly withrespect to the iris 653 and lens 655 of the eye. In FIGS. 39A and 39B, agenerally linear drug delivery implant 660 is disposed in a cross-linkedpocket in the peripheral portion of the cornea 652.

Turning to FIGS. 41A and 41B, it can be seen that an eye generallyincludes a lens 663, an iris 665, cornea 666, an anterior chamber 667, apupil 668, and a limbus 670. In FIGS. 42A and 42B, a one-partsemi-circular drug delivery implant 672 is shown disposed in across-linked pocket in the peripheral portion of the cornea 666. InFIGS. 43A and 43B, a doughnut-shaped or ring-shaped drug deliveryimplant 674 is shown disposed in a cross-linked pocket in the peripheralportion of the cornea 666. In FIGS. 44A and 44B, a generally linear drugdelivery implant 676 is shown disposed in a cross-linked pocket in theperipheral portion of the cornea 666.

In one or more embodiments, the implant is inserted in the cornealpocket through a small external incision made into the corneal pocket.

In one or more embodiments, the pocket itself can be filled withbiodegradable nanoparticles for drug delivery to the entire ocularstructures from the cornea to the optic nerve, and all tissues inbetween. The medication(s) can be anti-inflammatory, anti-infective,immune-suppressants, AntiVEGFs, biologics, Anti-PDGF, Anti IL-6, Rhokinase inhibitors, nerve growth factors, anti-glaucoma medications,gene(s) delivery in conjugation with viral or non-viral nanoparticles,such as nanoparticles, quantum dots, biodendrimers, etc. coated withpolyethylene glycol (PEG) or cell penetrating agents along with anantibody to the specific tissue. This permits the genes or medicationsto be delivered after their migration out of the implant and the cornealpocket and to attach to the targeted cells in the cornea, conjunctiva,trabecular meshwork, retinal ganglion cells or photoreceptors, retinaland optic glial or nerve cells or their axons etc.

In one or more embodiments, the one or more medications in the drugimplant may be anti-inflammatory agents, such as steroids,Dexamethasone, NSAIDS, Anti IL-17, Anti IL-6 and other Anti-ILs orantibiotics, fluoroquinolones, macrolides, cephalosporin A, vancomycin,aminoglycosides, penicillin and its derivatives or combination ofantibiotics, etc., anti-virals, ganciclovir, valcyclovir, etc.,anti-fungals, amphotericine B, etc., Anti-VEGFs, Avastin, lucentis,Aflilbercept, Anti-IL-6, anti-parasitic, etc., or otheranti-inflammatory agents, such as NSAIDs after any corneal surgery andact therapeutically to various diseases affecting the conjunctiva (e.g.,dry eye), immune-suppressants, such as cyclosporine A, Mycophenolicacid, anti-proliferative agents, anti-metabolite agents, in uveitis,choroiditis or other medications, such as anti-glaucoma medication orcombination of medications, gene delivery, DNA, RNA, siRNA etc. alongwith viral or non-viral delivery vehicles and CRISPR-cas9 mediatedhomology-independent targeted integration (HITI) or homology directedrepair (HDR) to modify the genetic components of various diseases of theeye.

In one or more embodiments, repeated crosslinking of the pocket can beperformed as needed to prevent new cellular ingrowth and adhesion aroundthe implant from the corneal tissue so that the implant's barrier ismaintained, and the implant can be removed or replaced as needed (e.g.,if the eye needs another or a combined medication to regulate diseaseprocess, such age related macular degeneration, glaucoma, uveitis,choroiditis or an infectious process of any origin).

In one or more embodiments, the peripheral cross-linked pocket is usedto insert or inject medications needed to treat a corneal disease orglaucoma or a disease of the posterior segment. The medication can be ina form of nanoparticles, microspheres, lipid coating or PEG,streptavidin, biotin coating, etc., micelles, liposomes, thermosensitivechitosans, etc.

In one or more embodiments, one can inject or implant in the peripheralcorneal pocket large-sized flexible, semi-solid or porous or solid rod,flat or tube or any shape and size polymeric material that can beabsorbed with time and the medication is released slowly to the corneaor the anterior chamber of the eye or diffuses to the back of thechoroid or retina and optic nerve.

In one or more embodiments, the diameter of these rod or flat-shapedshape implants can vary between 10 microns to 1 millimeter (mm) indiameter or larger with a length of 1 to 50 mm or longer.

In one or more embodiments, the porous tube can be made ofsemi-permeable non-biodegradable material that permits only thediffusion of the fluid/medication, etc. in and out of the tube,implanted in the peripheral cross-linked pocket. In these one or moreembodiments, the tube can be refilled with medication as needed. Forexample, as shown in FIG. 36, the implant 640 in the form of arectangular tube is refillable by injection with a needle 642.

In one or more embodiments, the drug implant tube contains stem cells,embryonic stem cells, ciliary hormone producing cells, or other hormoneor factors producing stem cells, neuronal or glial stem cells,Mesnchymal stem cells, trabecular meshwork stem cells, limbal stemcells, modified skin stem cells, etc. in a biocompatible fluid thatpermits nutrition to reach the cells injected in the tube where thecells are immortalized to produce one or the other medication, growthfactors, such as ciliary neurotrophic growth factor, RPE growth factor,nerve growth factors, anti-VEGFs, or other medications needed.

In one or more embodiments, the non-biodegradable tube with pores fordrug and cell delivery is implanted in a cross-linked pocket with animplant in any part of the body for medication and cell delivery forvarious medications and functions.

In one or more embodiments, the implant is coated with biocompatiblepolymer(s) that is used for delivery of stem cells with medication in acorneal pocket. The implant has larger diameter holes of 5 microns andmore in its wall permitting the cells to escape from the tubular implantinto any tissue (e.g., corneal pocket containing stem cells, embryonicstem cells, ciliary body factor producing stem cells, neuronal or glialstem cells, Mesenchymal stem cells, trabecular meshwork stem cells,Limbal stem cells, modified skin stem cells, etc. in a biocompatiblefluid) that permits nutrition to reach the cells injected in the tubewhere these cells can grow and pass through the holes of the implant andmove toward a tissue. In FIG. 32, the tubular implant 626 has smallholes 628 disposed in the circular peripheral side thereof, whereas thetubular implant 630 in FIG. 33 has large holes 632 disposed in thecircular peripheral side thereof.

In one or more embodiments, the implant contains stem cells, embryonicstem cells, cilliary body hormone producing stem cells, neuronal orglial stem cells, Mesenchymal stem cells, trabecular meshwork stemcells, limbal stem cells, modified skin stem cells, etc. in abiocompatible fluid that permits nutrition to reach the cells injectedin the tube along with Rho kinase inhibitors or Nerve growth factors tostimulate their regeneration and migration of theses cell and repair thepathology in the tissue.

In one or more embodiments, genetically modified cells are used toproduce needed enzymes and medications. The combination of cross-linkingof the cornea and killing the corneal cells and lack of vessels in thecorneal makes it a suitable place for these cells in the tube implant tosurvive inside the tube without being attacked by the cellular body'sresponse, thereby creating an immune privileged space for these cells tosurvive and produce medications needed locally or systemically (e.g. inmany genetic diseases of the cornea such as Fuchs dystrophy, etc.).

In one or more embodiments, the pocket can be filled with a polymericmaterial that can become more semisolid, or becomes a gel, and containsmedication for slow release of medication.

In one or more embodiments, the medication can be injected in theperipheral pocket along with corneal stem cells from the limbus orgenetically modified skin stem cells, embryonic or pluripotential stemcells, or mesenchymal stem cells grown in the culture for implantation,in cases of cell loss of endothelium, or in genetically caused cornealopacification, such as macular dystrophy or trauma.

In one or more embodiments, the stem cells are mesenchymal stem cellsinjected in the corneal pocket along with ROCK inhibitors to replace orrepair a cloudiness of the cornea.

In one or more embodiments, the stem cells are nerve cells to induceregeneration of the damaged corneal nerve (e.g., in diabetic patient)and after traumatic corneal injuries or after LASIK surgery.

In one or more embodiments, all tubular drug implants or devices arereplaceable with ease.

In one or more embodiments, the tube can be refilled with medication tobe used as slow release drug delivery that releases the drug in thecornea and anterior of the posterior segment of the eye.

In one or more embodiments, the tube is used for taking fluid samplesfrom the eye.

In one or more embodiments, one creates an intrastromal corneal pocketin the peripheral cornea involving 2-4 mm 4-8 mm in width areasinvolving the cornea and the anterior sclera after bleaching out theperipheral conjunctival capillaries with a low dose of vasoconstrictivemedication such as 0.5%-1% phenylephrine applied locally with a Q-tippedapplicator using a femtosecond laser.

In one or more embodiments, a small knife can be used to create a pocketin the cornea or elsewhere under the skin etc. if needed.

In one or more embodiments, the pocket width is more toward the cornealside than the scleral side or vice versa. The circumferential extent ofthe pocket can be 1 degree to 360 degrees of the corneal periphery (seeFIGS. 38A, 38B, 42A, 42B, 43A, and 43B).

In one or more embodiments, using a small incision to access theintrastromal incision, one uses a curved probe to separate the cornealadhesion for injection of a photosensitizer cross-linker, such asriboflavin, at the desired concentration of 0.5% -4% in a biocompatiblefluid, such as a physiological saline solution, etc. or suspension ofparticulates in a volume of 0.01 milliliters (ml) to 1 milliliter (mm)as needed only for the extent of the corneal pocket to cover theinternal walls of the pocket for a desired duration that thephotosensitizer penetrates at least 20 microns and beyond the cornealpocket in the corneal stroma to isolate that localized area of thecornea from the rest so that it does not respond with cell migrationinto the surrounding implant and so that it avoids tissue boundingtogether or to the implant.

In one or more embodiments, 0.01 ml to 0.1 ml of 0.02-2% lidocaine orbupivacaine solution can be injected alone or along with thephotosensitizer in the corneal pocket to anesthetize the cornea for thenext 1-15 hours, thereby eliminating pain sensation or discomfort of thesurgery, and dry eye after surgery.

In one or more embodiments, the width of the corneal pocket can be 1-3mm as needed. The peripheral corneal pocket can be circular,semi-circular, C-shaped, doughnut-shaped, straight, curved, or any othershape.

In one or more embodiments, the cross-linked pocket can be located at adesired distance from the Bowman's membrane in the corneal periphery.

In one or more embodiments, the ultraviolet (UV) radiation or otherappropriate wavelength of light at the desired power 0.5-50 mW/Cm2 andduration of 1-15 minutes, or other radiation with another wave length isapplied externally in a stationary pattern or as a continuouspainting/oscillatory technique with a focused small sized spot of 1-4 mmand a high energy to cover the width of the pocket, or on a paintingoscillatory fashion entering the corneal pocket with a small diameterfiber optic and to activate the photosensitizer in the corneal pocketand crosslink the collagen of the corneal stroma surrounding the cornealpocket, and preventing the wall from adhering to itself or to a futureimplant, while providing a physical stability to the wall of the cornealpocket and preventing cell migration and rejection of an implant.

In one or more embodiments, ultraviolet (UV) radiation at the desiredpower in a stationary or focused light for a duration of 10 seconds to15 minutes for the stationary radiation, or for a duration of 10 secondsto 20 minutes for the painting approach, depending on the power of theradiation and the length of the pocket used (or other radiation withanother photosensitizer and wave length) is applied externally or via afiber optic inserted inside the pocket to activate the photosensitizerand cross-link the collagen of the corneal stroma surrounding thecorneal pocket while preventing cell migration, encapsulation, orrejection of the implant and protecting the anterior corneal stroma andthe stem cells.

In one or more embodiments, the corneal pocket is three-dimensionallycut in order to remove a part of the stroma and create a space for theimplant.

In one or more embodiments, the wall of the corneal pocket can absorbthe photosensitizer from the implant after it is dipped in aphotosensitizer solution or the implant is coated with nanoparticles ofthe cross-linker and placed in the corneal pocket to leak out, which isthen followed by UV radiation at the desired power and duration or otherradiation with another wave length applied externally or internally viaa fiber optic to activate the photosensitizer in the corneal pocket andcross-link the collagen surrounding the implant. This technique providesa physical stability to the cornea preventing adhesion or gluing theimplant to the surrounding tissue and preventing fibrous ingrowth orencapsulation or rejection of the implant, which can lead to implantrejection. This makes it possible to exchange the implant when neededwithout much trauma to the cornea surrounding the implant.

In one or more embodiments, the photosensitizer is conjugated to thesurface of the implant having a polymeric coating, such as collagen,that releases the photosensitizer (e.g., riboflavin) from the implantonce it is exposed to the water content of the tissue in the cornealpocket surrounding it. The riboflavin is released and stains the wall ofthe implant which is subsequently cross-linked with UV light. Thisprevents tissue adhesion between the implant and the corneal tissue andmaintains a potential space between the corneal wall and the implant,thereby preventing activation of an immunologic response or neovasculartissue response by releasing from the tissue vascular endothelial cellfactors (VEGF) in response to a foreign implant. The cross-linkingprocess can be repeated as needed every 6 months to a year or more asneeded. The cross-linking of the collagen protects the implantcontaining particulate medication(s), which releases the drug for a longtime, and prevents the pocket from being invaded by the immune cellularelements and keeps the lumen of the tube shaped implant open.

In one or more embodiments, during the cross-linking, the corneal pocketremains pristine not allowing cell traffic or access to the pocketsurrounded by the cross-linked amorphous collagen or other cross-linkedtissues located in that area.

In one or more embodiments, the crosslinking can be repeated again inthe postoperative period after implantation by injecting a cross-linkerin the corneal pocket through a needle inside the wall of the pocket,which diffuses readily through the potential space around the implantand the wall of the pocket, and then is irradiated with UV light fromthe outside.

In one or more embodiments, the implant can be made of silicone,acrylic, methacrylate, HEMA, metallic or non-metallic, synthetic,organic, polymeric biodegradable, etc., coated with another or abiocompatible polymeric materials or a mixture thereof or coated with,for example, collagen or elastin, formed with a desired thickness of 2microns to 100 microns, and conjugated with a cross-linker or thecross-linker is injected in the potential pocket space in the tissue andis cross-linked.

In one or more embodiments, the implant is made by the use of 3-Dprinting technology with the desired material, shape, size or thickness,transparent or non-transparent organic or non-organic or a mixture ofthem, a material such as collagen elastin, synthetic polymers can becoated again with riboflavin nanoparticles with one or morebiocompatible polymer(s), and cross-linked with UV light prior to orpreferably after implantation.

In one or more embodiments, the implant is coated with a collagenpolymer to a desired thickness or in combination with another polymer,such as polyvinyl alcohol, chitosan, polycaprolactone, etc., conjugatedwith riboflavin or another cross-linker and cross-linked before or afterimplantation in a preformed pocket with an appropriate wavelength oflight or UV radiation to cross-link the polymeric coating inside thebody allowing the cross-linker to be released in the tissue, and thencross-link the tissue surrounding the implant in order to, afterimplantation, release the incorporated medication from the implantslowly without inciting cellular attraction or encapsulation of theimplant which inhibits a release of the medication(s) from the implantthat is unpredictable. For example, as shown in FIG. 30, the implant 618is coated with a polymer and/or a photosensitizer.

In one or more embodiments, the non-biodegradable flexible porous tubemade of polymeric material or a non-organic compound in combination withcross-linked organic polymer coating is filled with microspheres, drugnanoparticles incorporated in a polymeric material, such as polylacticglycolic acid, chitosan, liposomes, polycaprolactone, or lipid-coatednanoparticles, etc. containing the medication so as to release themedication slowly from the tube implant.

In one or more embodiments, the implant can serve as a reservoir thatreleases the medications though the pores of 1 to 3 microns in diameterin its wall, and then can be refilled repeatedly by injecting in thetubular implant the medication through a 33-34 gauge needle through thecornea surrounding the tube.

In one or more embodiments, the implant releases immunosuppressiveagents, such as cyclosporine, calcineurin inhibitors, mycophenolic acid,tacrolimus, siraliums, steroids, MPP inhibitors, NSAIDs,antimetabolytes, polycolonal antibodies, monocolonal antibodies, TNFinhibitors, Fingolimod, antibiotics, intraocular pressure (IOP) loweringagents, such as Rho kinase inhibitors, Fasudil, and other agents,pilocarpine, prostaglandin analogues, Brimonidine, etc., anti-virals,Anti-VEGFs, biologics, or neuroprotective releasing medication. Themedications being released as needed at concentrations of nanograms ormicrograms or mg/per hour depending on the polymeric material size ofthe holes, length of the polymer, etc.

In one or more embodiments, the implant can be positioned at any placein the body to control a function or release a medication without beingencapsulated by the surrounding tissue, due to the cross-linking of thepolymeric coating or the pocket being cross-linked prior to theimplantation, while the medication can be an anti-VEGF, neuroprotectiveagents, such as nerve growth factors, Rho kinase inhibitor such asFasudil, antibiotics, antiproliferative agents, anti-inflammatoryagents, etc. at a non-toxic, beneficial concentration.

In one or more embodiments, the implant is made using 3-D printingtechnology to the desired shape, size or thickness from any materialcoated with collagen, elastin, or made of collagen, elastin, etc. orsynthetic polymers which are further coated with more biocompatiblepolymer(s), such as acrylic, organic, etc., which are cross-linked priorto the implantation or coated with a cross-linker or the crosslinkingnanoparticles are done subsequent to its release in the tissue prior toradiation with the UV light. In another embodiment, the implant isformed from glass using 3-D printing technology (i.e., the implant is3-D printed glass).

In one or more embodiments, the implant is implanted in another part ofthe eye, such as under the conjunctiva, under the sclera, in the retinaor sub-retinal space, under the skin using an implant containingmedications such as Botox, or in other parts of the body using animplant which is coated with collagen to a desired thickness, dipped ina photosensitizer or has photosensitizer nanoparticles, such asriboflavin, etc. or the photosensitizer is injected surrounding theimplant and implanted in desired location, such as under or over thesclera in the choroid, under the conjunctiva, etc. Then, ultraviolet(UV) radiation or another wavelength of light is used to cross-link thetissue at the desired power and duration depending on what technique isused. In these conditions, a focused UV or another wavelength of lightis applied externally, in a painting oscillatory fashion only to thedesired areas or internally through a fiber optic, etc. to activate thephotosensitizer in the surrounding tissue where the implant is located.The cross-linked collagenous tissues surrounding the implant preventcreating an adhesion between the tissue and the implant or gluing thewall of the pocket together or to the implant. The cross-linkedcollagenous tissues surrounding the implant also have these additionalbenefits: (1) it is easier to replace the implant if needed, (2) fibrousingrowth or encapsulation is prevented, (3) it permits injection of thecross-linker again to repeat the cross-linking process if needed, and(4) it prevents rejection of the implant and contributes to the slowrelease of the medication from the implant. Also, these implants can actas a shunt for glaucoma, or drainage shunt for cerebrospinal fluid, orother part of the body, such as a bladder neck for urine if the drainagesystem is provided with a unilateral valve that only opens when thebladder pressure increases to certain level, etc.

In one or more embodiments, the injection of a small amount ofhyaluronic acid or other viscous fluid in the pocket simplifies theinserting of the implant in the peripheral corneal pocket or a pocketcreated in another tissue.

In one or more embodiments, the implant can be a biodegradable polymercarrying various medications and can be replaced.

In one or more embodiments, the implant is a tube-like structure havinga thickness or diameter of 0.02 millimeters (mm) to 0.4 millimeters (mm)in one direction and up to 8 mm in another (flat) width, and being 1-60mm long covering the entire corneal periphery without pressing thecorneal tissue in any direction. The implant may be filled with amedication(s), a fluid, or a combination of them.

In one or more embodiments, the tube is not biodegradable having holesmade in the wall of the tube with 0.2 to 3 microns in diameter, or 5microns to 500 microns in diameter, to permit diffusion of themedications or cells placed in it to produce desired needed proteins,hormones, nerve growth factors, or other products needed for other bodycell survival, such as cornea, retina, brain, etc.

In one or more embodiments, the tube has holes that are 5 to 15 micronsin diameter so as to permit stem cells to exit the tube. The tube can bebiodegradable implanted in a lightly cross-linked corneal pocketpermitting, for example, stem cells to proliferate and/or migrate to thecornea. The stem cells can be obtained from limbal stem cells ormesenchymal stem or skin and cultured cells prior to the injection inthe cornea or in another part of the body.

In one or more embodiments, the device is implanted in the wall of thevitreous cavity with one end closed and one end open to the vitreouscavity, or the implant can be under the retina or it can penetrate boththe retina and the choroid and permit release of medication or thecells.

In one or more embodiments, the implant is implanted in the tissuesurrounding the eye, on the face, etc. with one end closed and one endopen to the tissue. The implant can be removed after the drug isreleased, and then replaced.

In one or more embodiments, the repeated crosslinking of the tissuesurrounding the pocket can be performed as needed to prevent cellularingrowth, and the implant can be removed and replaced as needed (e.g. inage related macular degeneration) to maintain delivery of theanti-glaucoma medication, anti-VEGFs, immunosuppressive oranti-inflammatory agents, or nerve growth factors, or Rho kinaseinhibitors.

In one or more embodiments, the peripheral cross-linked pocket is usedto insert or inject medications needed to treat a corneal disease,glaucoma, or a disease of the posterior segment. The medication can bein a form of nanoparticles, microparticles, micelles, liposomes,chitosans, polycaprolactone as nanoparticles, dendrimers, etc.

In one or more embodiments, one can inject or insert an implant in theperipheral corneal pocket that is in the form of a large-sized flexible,semi-solid or solid, porous or solid rod-shaped implant, a flat implant,or tube-shaped implant that contains medication, or any shape and sizepolymeric material that can be absorbed with time and the medication isreleased slowly to the cornea or the anterior chamber of the eye ordiffuses through the anterior chamber or through the sclera to the backof the eye, for treatment of the choroidal or retina and optic nervediseases. As shown in FIGS. 31A-31C, the implant may be in the form of asolid implant 620, a porous implant 622, or a solid tubular implant 624with an open end. Also, as shown in FIG. 35, the implant may be in theform of a semi-solid or silicone tubular implant 636 with one closed end638 and one open end 639.

In one or more embodiments, the diameter of the rod or flat-shapedimplant can have a length of 1 microns to a few millimeters (mm), or thelength can be 1 to 40 millimeters (mm) or longer.

In one or more embodiments, the non-biodegradable tube is open-ended sothat the medication exits only from one or both ends of the tube.

In one or more embodiments, the medication can be released for aduration of from 3 months to 3 or more years, such as when containingnanoparticles of fluoroquinolone dexamethasone, diclofenac, etc., andthe implant can be replaced or removed if the desired effect has beenachieved or reinjected in the corneal pocket.

In one or more embodiments, the tube is closed ended, but has pores fordiffusion of the medication. For example, refer to the implants 626, 630in FIGS. 32 and 33.

In one or more embodiments, the implant can be placed near any joint inthe body and the cross-linking is done using ultraviolet (UV) radiationthrough the skin or through the fiber optic as described for localizeddrug delivery.

In one or more embodiments, the porous tube can be made of semipermeablenon-biodegradable material that permits only the diffusion offluid/medication, etc. in and out of the tube, and the tube is implantedin the peripheral cross-linked corneal pocket, wherein the tube can berefilled with medication as needed via an injection using a 33-34 gaugeneedle. For example, refer to FIG. 36.

In one or more embodiments, the tube contains cells in a biocompatiblefluid that permits nutrition to reach the cells which are injected inthe tube where the cells are immortalized to produce one or moremedications, growth factors, such as a ciliary neurotrophic growthfactor, RPE growth factor, nerve growth factors, anti-VEGFs, or othermedications needed.

In one or more embodiments, the implant contains genetically modifiedcells producing other needed enzymes and medications. The combination ofcrosslinking of the cornea produces a wall of amorphous, acellularcollagen and the corneal location that lacks vessels provides a suitableplace for these cells in the tube implant to survive and producemedications as needed, which otherwise would have to be given repeatedlyeither locally or systemically, and in many genetic diseases of thecornea, such as Fuchs dystrophy, the cells have to be injected in thesubconjunctival space where the cells could be attacked by the normalcellular body's immune response. The cross-linked pocket with theimplant creates an immune-privileged space in the cornea or elsewherefor these cells to survive. For example, refer to FIGS. 38A-45B.

In one or more embodiments, the medication in the implant can be in anyform or composition, such as antibiotics, anti-inflammatory, immunesuppressants, anti-glaucoma medication, anti-vascular proliferation,stimulatory, such as Rho inhibitors. The polymers can be made ofbio-degradable compounds, such as polylactic, polyglycolic acid or acombination of them, polycaprolactone, etc.

In one or more embodiments, the corneal cross-linked pocket contains atubular implant filled with particulate immunosuppressive agents, suchas cyclosporine etc., that release the medication at a constant, but lowconcentration of micrograms as needed. The medication diffuses in thecornea, sclera, and/or conjunctiva, thus eliminating the burningsensation of topical cyclosporine drops and the need for daily dropadmiration in dry eye syndromes, or after refractive surgery or otherdiseases.

In one or more embodiments, the medication can be injected in theperipheral pocket along with corneal stem cells taken from the limbus orgenetically modified stem cells and grown in the culture forimplantation.

In one or more embodiments, as shown in FIGS. 40A and 40B, anon-biodegradable implant tube 662 as described herein is implanted inthe cross-linked corneal pocket of the cornea 652 of the eye with iris653 and lens 655, and the implant tube 662 is connected to the anteriorchamber 657 with the aqueous fluid via a thin 23-34 gauge needle 664,where biomarkers such as VEGFs, glucose, and analytes, etc. are presentboth inside the aqueous and the tube system made of soft silicone.Similarly, as depicted in FIGS. 45A and 45B, an implant 678 is implantedin the cross-linked corneal pocket of the cornea 666 of the eye withiris 665, and the implant 678 is connected to the anterior chamber 667with the aqueous fluid via a needle 680. The implants 662, 678 can bepenetrated with a 30-34 gauge needle from outside and the aqueous can beaspirated in a volume of less than 0.50 microliters repeatedly over along period of time without causing a collapse of the anterior chamber.The volume of the anterior chamber is 25 times more than the samplefluid taken. The minimal amount of aqueous fluid withdrawn will bereplaced by the eye in less than 10 minutes. This provides a means ofobtaining easily a fluid biopsy repeatedly from the eye withoutpenetrating the entire cornea or the eye wall directly with thecomplication of iris or lens injury and retinal injury. The fluid samplecan be examined in chronic disease processes, such as uveitis forbiomarker of a disease, viral infection that persist in the eye longafter the body has healed, such as Ebola, Zika, Herpes viruses or otherviral diseases or non-viral infections that can be detected and treatedappropriately. The biomarkers can be obtained from the implanted tube,and can provide valuable information on many metabolic diseases of thebody or the eye, a systemic disease (e.g., Alzheimer disease), agerelated macular degeneration, glucose level, or other analytes (e.g.,diabetes) in diabetic retinopathy and other slow progressivedegenerative eye diseases, tumors, infection, uveitis, poisoning or drugoverdose, etc.

In one or more embodiments, a plurality of implants are implanted in thecornea of the eye. In these one or more embodiments, each of theimplants is used for a different purpose. For example, a first one ofthe implants may be in form of a corneal drug delivery implant used fordelivering one or more medications to the eye, as described above. Asecond one of the implants may be used for taking liquid biopsies from aportion of the eye, as described herein (e.g., extracting a liquidbiopsy of the aqueous fluid from the anterior chamber of the eye). Athird one of the implants may be used for stem cell delivery and/or genetherapy in the manner described above. A fourth one of the implants maybe used for measuring the intraocular pressure of the eye of the patient(e.g., intracorneal implant comprising a pressure sensor). That is, thefourth implant may contain a pressure sensor configured to measure anintraocular pressure of an eye and to output a signal based on themeasured intraocular pressure of the eye, the pressure sensor configuredto be implanted in a cornea of the eye; a processor operatively coupledto the pressure sensor, the processor configured to generate intraocularpressure data based upon the signal outputted by the pressure sensor;and a transmitter device operatively coupled to the processor, thetransmitter device configured to transmit the intraocular pressure datagenerated by the processor to a remote receiver located outside of theeye, the transmitter device configured to be implanted in the cornea ofthe eye. In addition to the pressure sensor, the third implant mayfurther comprise a needle configured to penetrate a posterior portion ofthe cornea of the eye, the needle configured to open into the anteriorchamber of the eye so as to measure the intraocular pressure of the eyewithout obstructing vision through the central cornea.

In one or more embodiments, one can measure the amount of VEGF presentin the aqueous providing information on the disease progressionrequiring treatment (e.g., anti-VEGFs or no treatment). Anti-VEGFs oranother medication can be administered directly in the tube to reach theposterior segment avoiding repeated intraocular injection through thesclera, without having the risk of retinal detachment or lens injury. Asanother example, liquid biopsy of aqueous in a patient with diabeticretinopathy, where the retina is in need of treatment with the lasercoagulation, provides the information regarding whether the diseaseprocess is under the control or not.

In one or more embodiments, for the first time one can obtain from theaqueous biopsy, instant information needed for the doctor to diagnose adisease process at the bedside and be able to follow the process over along period of time with ease.

In one or more embodiments, nanoparticles carrying other medications canbe delivered as slow release nanoparticles from the tube in the anteriorchamber to treat glaucoma for a long period of time, thereby eliminatingthe need for repeat therapy. These medications may include pilocarpine,prostaglandin analogues for treatment of glaucoma, Rho kinaseinhibitors, or neuroprotective agents or Brimonidine, etc.

In one or more embodiments, the implanted tube is filled with desiredmedications, as described above, and is coated with collagen or albuminloaded with riboflavin particles that are diffused after implantation inthe pocket. The ultraviolet (UV) radiation used for cross-linkingpermits the diffusing of the medication from the implant as a slowrelease device, and prevents vascular growth around the implantcontaining the medication.

In one or more embodiments, the implanted tube can be 100 microns to 1millimeters (mm) in diameter and 4 mm to 40 mm long, or less than 100micron in diameter and no longer than a few millimeters in length. Theimplanted tube maybe filled with any desired medication to be implantedin any tissue and cross-linked after implantation.

Methods disclosed hereinafter include administering Wnt inhibitorseither alone, or in combination with Rho inhibitors, or one or more Wntinhibitors with the dendrimers or liposomes, or (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD that are useful for alleviating the effects of conditions thatare caused by acute or chronic inflammatory processes, such as chronicinflammatory dry eye disease, diabetes, optic nerve neuritis, scleritis,keratitis, chronic Meibomian gland inflammation, and uveitis.

In one embodiment, Wnt inhibitors or Rho kinase inhibitors with thedendrimers or liposomes, or as nano- or microparticles with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD are used as topical drops, spray,ointment, gel, or a non-toxic injectable formulation under theconjunctiva, in the corneal pocket, or in the vitreous to treat the dryeye syndrome or mucosal inflammatory diseases, such as uveitis, opticnerve neuritis, diabetic macular edema, cystoid macular edema aftercataract surgery, macular edema in uveitis, vasculitis, Behçet'sdisease, sarcoidosis, multiple sclerosis, lichen planus, chronic jointdisease arthritis, chronic choroiditis, plantar fasciitis, parsplanitis, scleritis, iritis, and/or scleritis gingivitis.

A method of treating dry eye with deficiency of aqueous production whichis associated often with the Meibomian gland disease, affecting about 7%to 34% of all Americans, pathophysiology of chronic dry eye diseaseincluding a cycle of inflammation involving both innate and adaptiveimmune responses is also disclosed herein.

In one embodiment, dry eye syndrome (DES) or keratoconjunctivitis sicca,a disease affecting tear production leading to damage to the cornealsurface, associated often with disturbance of Meibomian gland, lachrymalgland, conjunctival goblet cells, nasolacrimal duct and pain sensationis treated by Wnt inhibitors or Rho kinase inhibitors used as topicaldrops, ointment, gel, non-toxic injectable formulation such as nano- ormicroparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, the method used for treatment of the eye utilizesover the counter physiological saline solutions with some othercomponents to affect the inflammatory component of the dry eye orimprove on the composing of the tear film, such as tear film osmolarity,or adding lipids, mucin, etc. Other topical medication includeTheraTears® (Advanced Vision Research), Refresh® and Celluvisce®(Allergan), Tears Natural® and Bion Tears® (Alcon), GenTeal® andHypoTears® (CIBA Vision), each of which contain electrolytes and hasvarying pH levels, osmolarities, Restasis® (0.05% cyclosporine,Allergan),), and Xiidra® (5% lifitegrast, Shire), which attacks theinflammatory process by a different mechanism than cyclosporine. Most ofthese medications are applied as a drop to maintain the conjunctivalwetness as needed usually 1-3 drops during the day or ointment at nightmost of these medications may be used in combination with Rockinhibitors, such as Fasudil, or Wnt inhibitors.

In one embodiment, the Rock inhibitors not only reestablishes the tearproduction by reducing the conjunctival inflammatory cytokines andinflammatory response, but enhances the nerve fibers to grow andreestablish the function of conjunctival goblet cells to produce mucin,which is essential for tear film lubrication. RHO associated proteinKinase (Rock) is a kinase belonging to the family of serine-threonineKinase involved in regulating the shape and the cytoskeleton of thecells, it is an important regulator of cell migration, stimulates PTENphosphatase activity, leading to uncontrolled cell division in cancer.Rock is active in inflammatory processes, cancer, diabetes, and manyneurodegenerative diseases such as dry or wet form of age relatedmacular degeneration. Therefore, Rock inhibitors inhibit inflammatoryprocesses.

In one embodiment, Rock inhibitors may be used in combination withfunctionalized nanoparticles of polycaprolactone, polylactic orpolyglycolic acid, as nano- or microparticles with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD etc. to reduce the inflammation during immune therapy orthermoimmune therapy.

In one embodiment, a potent ROCK inhibitor administered systemically,locally, as an implant, orally, topically as functionalizednanoparticles, microparticles, dendrimers or such as bioavailableFasudil hydrochloride, inhibitor of cyclic nucleotide dependent- andRho-kinases GSK 269962 is used. In one embodiment, potent and selectiveROCK inhibitor GSK 429286, Selective Rho-kinase (ROCK) inhibitor H1152dihydrochloride, or Botox, Wnt inhibitors, and brimonidine are used asan implant for slow release of the medication.

In one embodiment, selective Rho-kinase (ROCK) inhibitor Glycyl H 1152dihydrochloride, or another selective Rho-kinase (ROCK) inhibitor isadministered as a topical ointment, drop, or gel. Also, a more selectiveanalogue of H1152, that is cell-permeable, a selective Rho-kinaseinhibitor OXA 06 dihydrochloride, a potent ROCK inhibitor PKI1447dihydrochloride, potent and selective ROCK inhibitor antitumor SB772077B, a potent Rho-kinase inhibitor, vasodilator SR 3677dihydrochloride, a potent, selective Rho-kinase (ROCK) inhibitorTC-S7001, a potent and highly selective ROCK inhibitor, orally activeY-27632 dihydrochloride or Botox also may be administered as nano- ormicroparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, ROCK inhibitors include, but are not limited to,Fasudil, Ripasudil, RK1-1447, Y-27632, GSK429286A, Y-30141, etc. Theymay be administered in a polymer, an implant, microparticles ornanoparticles, or porous silicone implant, etc. They may also containpoly(amidoamine) (PAMAM), poly(amidoamine-organosilicon) (PAMAMOS),poly(propyleneimine) (PPIO), poly(caprolactone), poly(lactic acid)(PLA), polylactic-co-glycolic acid (PLGA); may be tecto, multilingual,chiral, hybrid, amphiphilic, micellar, multiple antipen peptide, andFrechet-type dendrimers; may be functionalized microparticles ornanoparticles with an antibody and/or a ligand for a receptor orcovalent coupling to one or more of cell penetrating peptides (CPP),arginine-CPP, cysteine-CPP, polyethylene glycol (PEG),biotin-streptavadin, and/or acetyl cysteine or as nano- ormicroparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, ROCK inhibitors include, but are not limited to,Fasudil, Ripasudil, RK1-1447, Y-27632, GSK429286A, Y-30141, etc. Theymay be administered in a suspension, or solution over the cornea orinjected inside the corneal channel provided with a femtosecond laser asnano- or microparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, the implant may be a C-shaped or ring-shaped flexibleimplant with a diameter of the implant of 20 microns to 200 microns ormore, with a length of 1-12 mm and if it is circular with the diameterof 3-8 mm or less than the diameter of the lens capsule after cataractextraction without exerting any pressure or tension on the lens capsuleor expanding it. In one embodiment, the polymeric material can be orporous silicone implant etc. They may also contain poly(amidoamine)(PAMAM), poly(amidoamine-organosilicon) (PAMAMOS), poly(propyleneimine)(PPIO), poly(caprolactone), poly(lactic acid) (PLA),polylactic-co-glycolic acid (PLGA); may be tecto, multilingual, chiral,hybrid, amphiphilic, micellar, multiple antipen peptide, andFrechet-type dendrimers; may be functionalized microparticles ornanoparticles with an antibody and/or a ligand for a receptor orcovalent coupling to one or more of cell penetrating peptides (CPP),arginine-CPP, cysteine-CPP, polyethylene glycol (PEG),biotin-streptavadin, and/or acetyl cysteine or as nano- ormicroparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, the implant is injected in the cornea or scleralpocket using a 20 gauge or 25 gauge or 27 gauge or 30 gauge needle.

In one embodiment, one or more corneal pockets 720 are produced in thecorneal stroma 712 of an eye 710 with a femtosecond laser or a smallknife (see FIG. 46) or one or more scleral pockets 718 are formed in thesclera 714 of the eye 710 or under the conjunctiva and the implant 724carrying Rho inhibitors, Wnt inhibitors, Fingolimod, or antibiotics, isplaced in the pocket 718 or 720. As shown in FIG. 46, a subconjunctivalimplant 722 may also be implanted in the eye 710 with optic nerve 716.In another embodiment, a femtosecond laser flap or a flap formed by aknife is created in the corneal stroma of an eye 726 as a LASIK flap 732and after correcting the refractive error either with an excimer laseror an inlay, then an implant 734 is placed under the corneal flap 732(see FIGS. 47-48). As shown in FIGS. 47 and 48, the eye 726 with lens728, iris 730, and optic nerve 736 comprises a corneal drug deliveryimplant 734 disposed in the stromal tissue underneath the LASIK flap732. FIG. 48 illustrates the eye 726 after the flap 732 has beenreplaced. In FIG. 48, the attached area of the flap 732 is representedby the dashed line 738, while the flap incision is represented by thesolid line 740. In yet another embodiment, a corneal pocket is createdwith femtosecond laser and an inlay is placed inside the pocket tocorrect refractive error of the eye followed with implantation of animplant in the pocket for drug delivery and the implant carrying Rhoinhibitors, Wnt inhibitors, Fingolimod, or antibiotics as nano- ormicroparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD), etc.

In another embodiment, after refractive surgery, the polymericmedication is applied either as a drop 746 or spraying to the cornea 744of eye 726 with sclera 742 (see FIG. 49). In yet another embodiment,after refractive surgery or traumatic corneal injury, a soft polymericcontact lens 748 carrying medication is placed over the cornea 744 toprotect it and treat it with medication (see FIG. 50), and the implantcarrying Rho inhibitors, Wnt inhibitors, Fingolimod, or antibiotics,Fasudil, brimonidine, etc. with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD).

In another embodiment, the polymeric drug delivery implant 760 has aC-shape or has a ring or a curved shape and is placed in the lenscapsule 754 of an eye 750 without expanding or putting tension orpressure on the capsule and the implant 760 carrying Rho inhibitors,Fasudil, or Wnt inhibitors, Fingolimod, or antibiotics, an agent tolower the intraocular pressure (IOP), NSAIDs, with as nano- ormicroparticles and/or with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD, etc. Thepolymeric implant 760 can be placed, either before or after theintraocular lens (IOL) implantation. The drug delivery implant 760 nevercovers the view of the patient (see FIG. 51). In FIG. 51, it can be seenthat eye 750 with lens zonules 752, lens capsule 754, iris 756, andcornea 758 comprises an intracapsular C-shaped or ring-shaped drugdelivery implant 760 that has been inserted into the lens capsule 754after cataract surgery on the eye 750.

In one embodiment, an implant 766 (e.g., a C-shaped or ring-shaped drugdelivery implant 766) is placed over the lens capsule 754 before thecrystalline lens is removed or can be placed on the lens zonules 752after cataract extraction and intraocular (IOL) lens 764 implantation(see FIG. 52), and the implant carrying Rho inhibitors, or Wntinhibitors, or Fingolimod, or antibiotics or an agent to lower IOP, suchas prostaglandin analogues, NSAIDs, etc.). In FIG. 52, it can be seenthat eye 750 with lens zonules 752, lens capsule 754, iris 756, opticnerve 762, and retina 768 comprises an intracapsular C-shaped orring-shaped drug delivery implant 766 disposed over the lens zonules752.

In one embodiment, the polymeric material is administered to thevitreous before or after removal of the vitreous for both visualizationand delivery of medication to the eye, and the implant carrying eitherRho inhibitors, or Wnt inhibitors, or Fingolimod, or antibiotics,NSAIDs, etc. as nano- or microparticles with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD).

In one embodiment, the polymeric drug delivery carrier can benanoparticles or microparticles or up to one mm in diameter injected asnano- or microparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) compound inthe eye cavity or in the pocket created in the cornea or sclera or underthe conjunctiva to place an implant. For example, as shown in FIG. 53,nanoparticles or microparticles 774 carrying one or more medications maybe injected into the vitreous cavity 770 of an eye 750 using a needle772. In FIG. 53, it can be seen that eye 750 with lens capsule 754, iris756, cornea 758, optic nerve 762, and retina 768 is being treated withnanoparticles or microparticles 774 carrying one or more medicationsthat are injected into the vitreous cavity 770.

In one embodiment, the implant is visible by the nature of themedication it carries or may be transplanted such as implants (if morethan one implant is provided) and comprises one or more medicationsselected from the group consisting of immunosuppressive agents,calcineurin inhibitors, mycophenolic acid, tacrolimus, siraliums,steroids, MPP inhibitors, NSAIDs, antimetabolytes, polycolonalantibodies, monocolonal antibodies, TNF inhibitors, Rho inhibitors, Wntinhibitors, Fingolimod, antibiotics, Brimonidine an intraocular pressure(TOP) lowering agents, pilocarpine, prostaglandin analogues,anti-virals, anti-VEGFs, biologics, neuroprotective medications, andcombinations thereof as a preparation to penetrate the tissue and thecells using as nano- or microparticles with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD).

In one embodiment, aqueous tear-deficient dry eye, occurring as a resultof not enough tears being produced due to a dysfunction of the lacrimalglands, is treated with Wnt inhibitors or Rho kinase inhibitors astopical drops, ointment, gel, or a non-toxic injectable formulations asnano- or microparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) for tissueand cell penetration and slow release.

In one embodiment, the Wnt inhibitors compound that is used includesFH535, IWP-2, PNU-74654, IWR-lendo, IWR-exo, demethoxycurcumin,sulforaphane and vitamin D, CCTO36477, KY02111, WAY-316606, SFRP, IWP,LGK974, C59, Ant1.4Br/Ant 1.4Cl, ivermectin, niclosamide, apicularen andbafilomycin, XAV939, XAV939, G007-LK and G244-LM, NSC668036, SB-216763,gemtuzumab, and akinumab as nano- or microparticles with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD because of their hydrophilic andhydrophobic nature.

In one embodiment, patients with moderate-to-severe dry eye having bothelements of evaporative dry eye and aqueous tear-deficient dry eye, andthat are on topical medications for other diseases, such as glaucoma,drops, or antibiotics containing preservative that over time damage theconjunctival goblet cells and other cells and induce dry eye syndrome,are treated with Wnt inhibitors or Rho kinase inhibitors as topicaldrops, ointment, gel, or a non-toxic injectable formulation or as nano-or microparticles with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) to releasemedication slowly without causing irritation of the conjunctiva or thecornea.

In one embodiment, administration of Wnt inhibitors, suchdemethoxycurcumin, sulforaphane and vitamin D, or Rho kinase inhibitors,such as Fasudil derivatives, is done as topical drops, a gel, anon-toxic injectable formulation, or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) or injectable Botox, 1-100 units asneeded, administered locally at multiple locations with the sameformulation having nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD or Rock inhibitors molecules atdoses of 1 picogram (pg) to nanograms to a few micrograms as a slowrelease delivery system.

In one embodiment, patients who are on topical medications for otherdiseases, such as glaucoma, drops or antibiotics containingpreservatives and over time damage the conjunctival goblet cells andother cells and induce dry eye syndrome, or patients with dry eye andglaucoma are treated either by implanting matrices polylactic acid orpoly glycolic acid, polyanhydride, or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) or chitosan polymers under theconjunctiva with slow release polymers containing either Wnt inhibitorsor Rock inhibitors, such as Botox or Fasudil derivatives, releasing themedication over months or years locally at multiple locations to releasethe non-toxic doses of the medications from 1 picogram (pg) to 1nanograms (ng) or more each day.

In one embodiment, patients who develop dry eye as a result of systemicmedication, such as in cancer patients developing dry eye afteradministration of checkpoint inhibitors in cancer immune therapy, aretreated either by Wnt inhibitors or Rock inhibitors with slow releasepolymers containing either Wnt inhibitors, such as demethoxycurcumin,CCTO36477, KY02111, WAY-316606, SFRP, IWP, LGK974, C59, Ant1.4Br/Ant1.4Cl, nitazoxanide (NTZ) ivermectin, niclosamide, sulforaphane andvitamin D, or Rock inhibitors, such as Botox or Fasudil derivatives,etc., releasing the medication over months or years locally at multiplelocations to release the non-toxic doses slow release medications from 1picogram to 10 nanograms each day or more using nano- or microparticleswith (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, the Sjorgen syndrome is associated with low salivaryflow, lymphocytic infiltration of the lacrimal gland and salivary glandauto antibodies in serum, rheumatoid factor, connective tissue diseases,such as Sjogren's syndrome, to the list of immune-related adverse eventsthat can develop during cancer treatment with immune checkpointinhibitors are treated with Rock inhibitors and Wnt inhibitors atnon-toxic concentrations of sulforaphane and vitamin D, CCTO36477,KY02111, WAY-316606, SFRP, IWP, LGK974, C59, Ant1.4Br/Ant 1.4Cl,nitazoxanide (NTZ), ivermectin, niclosamide, or Rock inhibitors such asBotox or Fasudil etc., releasing the medication slowly over months oryears locally at multiple locations to release the non-toxic doses slowrelease medications from 1 pg to 10 ng each day locally using as nano-or microparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) for slowrelease and tissue penetration.

In one embodiment, the patients being treated have a dry eye syndromeunassociated with SS (non-SS KCS) with a sensation of foreign body inthe eyes, photophobia, excessive tearing, ocular irritation and pain.Other symptoms are increased tear film osmolality, decrease in tearbreakup time, increase in the conjunctival enzymes metalloproteinase 9and 17, and changes in impression cytology of the conjunctival cells.These patients are treated with Rock inhibitors, such as injectableBotox, 1-10 units or in combination with metalloproteinase inhibitorsdoxycycline, low molecular with weight heparin, lovenox, anddexamethasone at concentration of 0.1%-5% as polymeric implant or asnano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, when inflammation is one of the mechanisms thatcauses damage to the ocular surface in dry eye disease seen inautoimmune diseases such as Sjögren's syndrome, and rheumatoid arthritisand neuropathic disorders, optic nerve neuritis, papillitis, scleritis,uveitis, inflammatory, infectious, chemical, traumatic diseases, etc.,the patients are treated with injectable Rock inhibitors, such as Botoxor Fasudil derivatives, conjugated with slow release polymer, such asporous silicone, etc. releasing the medication over months or yearslocally at multiple locations as the non-toxic doses slow releasemedications months to a year at concentration of 1 picogram to 10nanograms each day.

In one embodiment, the pathological conditions resulting in dry eyeinclude pemphigus and Sjogren's syndrome, which affect the eye by eitherdamaging the conjunctival cells responsible for maintaining the wetnessof the cornea and the conjunctiva, or by damaging the lacrimal glands ofthe eye and/or the meibomian glands of the eye lid or other pathologicalconditions resulting in dry eye include hypolacrimation, alacrima,Stevens-Johnson syndrome, marginal blepharitis pemphigus, ocularpemphigoid, scleritis, or diabetes are treated with Rock inhibitorFasudil, Botox, etc. at a picogram to nanogram concentration or incombination with metalloproteinase inhibitors, doxycycline 0.1%-5%solution, low molecular weight heparin 0.1%-5% solution, ordexamethasone 0.1-2% solution in combination with MTOR inhibitors at0.1%-5% solution or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) solution or suspension.

In one embodiment, the dry eye of patients occurring in post-cornealsurgery (including but not limited to post-LASIK surgery or PRK) withsurgical damage to the corneal nerves, other conditions resulting in dryeye including the aging process, environmental factors (e.g., dry homeand/or work environments), and extended use of visual display terminals(e.g., employment, recreation) are treated with Rock inhibitors, or incombination with metalloproteinase inhibitors, low molecular withheparin, or Wnt inhibitors or Rock inhibitor, such as Botox, 1-100 unitsadministered locally at multiple locations, small doses or Rockinhibitors molecule, Fasudil and its derivatives, etc., at doses of 1nanogram (ng) to a few micrograms (μg) as slow release polymer or asnano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, the dry eye can also occur after cataract surgery andrefractive surgery (i.e., the LASIK procedure or PRK) andphotorefractive keratectomy, smile procedure, partial or completecorneal transplants, which are the majorities of present refractivesurgery where these procedures are performed, but dry eye is more commonwith LASIK where the superficial nerves are cut, and where the eye driesout because the corneal reflex is affected and the eye subsequent tothese surgeries becomes dry while many eyes experience regeneration ofthe nerves, but it takes about one year or more to achieve it all.Patients with these conditions are treated with Rock inhibitors, or incombination with metalloproteinase inhibitors, low molecular withheparin, or Wnt inhibitors or Rock inhibitors, such as Botox, 1-100units administered locally over the cornea as drops 1-4 times daily orinjectable preparation at multiple locations, small doses or Rockinhibitors molecule, such as Fasudil or its derivatives, etc., at dosesof 1 nanogram or a few micrograms as slow release non-toxic preparationor as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, patients with paresis or paralysis of the fifth orseventh cranial nerves causing dry eye as a result of interfering withproper lid closure are treated with Rock inhibitors, such as Botox, 1-10units administered topically over the cornea at multiple locations,small doses or Rock inhibitors molecule Fasudil, or its derivatives,etc. at doses of 1 nanogram to a few micrograms as topical ointment,drop, gel, etc.

In one embodiment, Wnt signaling is involved in the control of stem cellproliferation. Wnt mutation causes developmental defects in many diseaseprocesses including inflammation and cancer, wet form of age relatedmacular degeneration, laser surgery, diabetic macular edema retinalvascular occlusion.

In one embodiment, the Wnt inhibitors compounds used are: FH535, IWP-2,PNU-74654, IWR-lendo. IWR-exo, nitazoxanide (NTZ), demethoxycurcumin,sulforaphane and vitamin D, CCTO36477, KY02111, WAY-316606, SFRP, IWP,LGK974, C59, Ant1.4Br/Ant 1.4Cl, Ivermectin, Niclosamide, apicularen andbafilomycin, XAV939, XAV939, G007-LK and G244-LM, NSC668036, SB-216763,gemtuzumab, akinumab Wnt inhibitors administered as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, the oral doses for the Wnt inhibitor niclosamide is 1to 2 g tablet once, or to repeat in 7 days, if needed.

In one embodiment, the small molecule Fasudil, a rock inhibitor Fasudil(HA-1077), a selective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632,small molecule inhibitor of ROCK1 and ROCK2, Botulinum toxin a is rockinhibitor marketed under the brand names Botox, Dysport, Myobloc,Xeomin, etc. Botulinum toxin, all having good penetration into thecornea, and do not increase intraocular pressure or cause cataracts andmay be dissolved in an organic solvent such as DMSO or alcohol orsterol, lanosterol, squalene, and/or squalamine, or containing apolyanhydride, poly(glycolic) acid, poly(lactic) acid, orpolycaprolactone polymer or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) for the treatment of the lid,conjunctiva, lacrimal gland corneal diseases and glaucoma.

In one embodiment, Fasudil is used as a single, oral 40-80 milligram(mg) dose orally as two 40 mg Fasudil tablets are administered.

In one embodiment, the methods include administering Wnt inhibitors,either alone or in combination with Rho inhibitors, orally, locally byinjection or drops, spray or ointment for alleviating the effects ofconditions that result in lack of moisture or wetness in the eye.

In one embodiment, Rho inhibitors, may be administered orally, locallyby injection or drops, spray or ointment for alleviating the effects ofconditions that result in lack of moisture or wetness in the eye, suchas the inflammatory conditions resulting in dry eye including pemphigusand Sjogren's syndrome, which affect the eye by either damaging theconjunctival cells, or by damaging the lacrimal glands of the eye and/orthe meibomian glands of the eye lid.

In one embodiment, the required treatment of Rho inhibitors such asBotox in 1-2 units, may be administered locally by injection or drops,spray or ointment or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD for inflammatory processes resultingin dry eye including hypolacrimation, alacrima, xerophthalmia,Stevens-Johnson syndrome, pemphigus, ocular pemphigoid, marginalblepharitis, nerve pain, diabetes, and/or post-corneal surgery aftercutting the corneal nerves (including but not limited to post-LASIKsurgery or PRK or corneal inlay). Other conditions resulting in dry eyeinclude the aging process, environmental factors (e.g., dry home and/orwork environments), and extended use of visual display terminals (e.g.,employment, recreation, etc.).

In one embodiment, the required treatment of Rho inhibitors, such asBotox in 1-2 units, may be administered locally by injection or drops,spray or ointment or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD for chronic inflammatory processes,lichen planus, arthritis, psoriasis, and plantar fasciitis.

In one embodiment, inhibition of Wnt signaling or ABC transporters byRNA interference may be a valuable therapeutic strategy in dry eyeincluding hypolacrimation, alacrima, xerophthalmia, and Stevens-Johnsonsyndrome, pemphigus, where the Wnt inhibitors administered as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, a number of Rock inhibitors are used in non-toxicdoses in combination with functionalized nanoparticles, conjugated withpolymeric coating, such as chitosan, polyanhydride, cyclodextrin as apotent ROCK inhibitor; bioavailable Fasudil hydrochloride, inhibitor ofcyclic nucleotide dependent- and Rho-kinases GSK 269962, potent andselective ROCK inhibitor GSK 429286, selective Rho-kinase (ROCK)inhibitor H1152 dihydrochloride, selective Rho-kinase (ROCK) inhibitorGlycyl H 1152 dihydrochloride, selective Rho-kinase (ROCK) inhibitor,more selective analogue of H1152, Cell-permeable, selective Rho-kinaseinhibitor OXA 06 dihydrochloride, potent ROCK inhibitor PKI1447dihydrochloride, potent and selective ROCK inhibitor, antitumor SB772077B, potent Rho-kinase inhibitor, vasodilator SR 3677dihydrochloride, potent, selective Rho-kinase (ROCK) inhibitor TC-S7001,potent and highly selective ROCK inhibitor, orally active Y-27632dihydrochloride and may be dissolved in an organic solvent such as DMSOor alcohol or sterol, lanosterol, squalene, and/or squalamine, or innano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin, orhydroxypropyl-b-cyclodextrin (bHPCD) or containing a polyanhydride,poly(glycolic) acid, poly(lactic) acid, or polycaprolactone polymer toreduce the inflammation processes in the eye, retina, choroid, sclera,lid, conjunctiva, or other mucosal diseases, or in the mouth.

In one embodiment, small molecule Wnt inhibitor PKF118-310, theWnt/β-catenin pathway inhibitor and Fasudil, a rock inhibitor Fasudil(HA-1077), a selective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632,small molecule inhibitor of ROCK1 and ROCK2, etc. may be dissolved in anorganic solvent such as DMSO or alcohol or sterol, lanosterol, squalene,and/or squalamine, or containing a polyanhydride, poly(glycolic) acid,poly(lactic) acid, or as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) or polycaprolactone polymer torelease non-toxic medication slowly at desired concentration to the eye.

In one embodiment, early management includes, the use of lubricants,artificial tear substitutes, ointment, gel, or emulsion. Topicalanti-inflammatory agents, topical rock inhibitors, anti-interleukin(IL1) TNF-alfa TNF-α, hyaluronic acid, low molecular heparin 0.1-5%solution alone or in combination with, metalloproteinase inhibitorsdoxycycline 0.1-5% solution immunosuppressive agent or inhibitormycophenylic acid as local or systemic therapy.

In one embodiment, topical Rock inhibitors are applied to the cornea asdrops or spray or subconjunctival injection as a slow release compoundcombined with chitosans or in nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD in 0.1 microgram/ml to 40microgram/ml or more for topical application.

In another embodiment, the Rock inhibitors are coated with the slowrelease polymers, such as lactic acid and/or glycolic acid at aconcentration of 200 nanograms to 1 micrograms/ml or more andadministered, topically, subconjunctival or inside the eyesubcutaneously inside the plantar fascia in the joint, etc. using nano-or microparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) for slowrelease and tissue penetration with or without cell penetrating agents.

In another embodiment, the Rock inhibitors are released from a polymericexplant or implant either placed over or under the conjunctiva andsutured to the sclera to release, e.g., Fasudil, etc. at concentrationsof 0.01 microgram/ml to 40.0 microgram/ml or more per day.

In one embodiment, the Rock inhibitors release, after placement in theupper or lower cul-de-sack of the conjunctiva or as a slow releasepunctal plaque or implanted subconjunctivally, at a rate of 1 picogramto a 10 nanograms/day of the medication or delivered as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) injection.

In one embodiment, the Rock inhibitors release, after placement of animplant or as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) in the suprachoroidal space, insidethe eye, behind the eye, inside the gingiva, subcutaneously in plantarfascia, or as a slow release polymeric plaque or implanted to releasemedication at a rate of 1 picogram to a 10 nanograms/day of thenon-toxic medication.

In another embodiment, the nanoparticles or dendrimers are conjugatedwith Rock inhibitors and chitosan delivered as a slow release systemthat can be released as a temperature sensitive polymer that melts at42-43 degrees used with a warm compressor over or under the lid, orlight thermal application, or the use of a compressive focusedultrasound applied to lid, conjunctiva or cornea or the lid releasing 1picogram to a 10 nanograms/day of the medication.

In one embodiment, the Rock inhibitors or Wnt inhibitors are deliveredas nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) with simultaneous application ofamniotic membrane and slow release nanoparticles applied post cornealsurgery, such as LASIK, or corneal inlay cataract corneal transplant, orcataract surgery and lens implantation or any other corneal surgicalintervention, scleritis, retinitis, vitreoretinal surgery, diabeticretinopathy, macular edema, at 10 picograms to 20 nanograms ofmedication per day.

In one embodiment, the Rock inhibitors or Wnt inhibitors are deliveredwith simultaneous application of amniotic membrane and low molecularweight heparin slow release nanoparticles applied post corneal surgery,such as LASIK, cataract, or cataract surgery and lens implantation,after retinal surgery, vitreous surgery, corneal transplant uveitisscleritis or chemical injury to the cornea or conjunctiva atconcentrations of 0.001 micrograms/ml to 40 micrograms/ml or more ortopical or subconjunctival Botox, at 1-100 units or topical at 1-5 unitsor more in a physiological solution of Botox, or similar preparations.

In one embodiment, the Rock inhibitors or Wnt inhibitors are deliveredwith simultaneous application of low molecular weight heparin (levonox)with other medications, such as tetracycline, Doxycycline ormetalloproteinase inhibitors, dexamethasone 0.1% -1% concentration asslow release polymeric nanoparticles or liposomes applied post cornealsurgery such as LASIK, PRK, cataract, corneal transplant, uveitis,scleritis or thermal or chemical injury to the cornea or conjunctiva,e.g., Fasudil derivatives, etc., at 0.1 micrograms/ml to 40micrograms/ml or more, or Botox at 1-3 units as nano- or microparticleswith (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) solution orsuspension.

In one embodiment, after LASIK or cataract surgery and lens implantationor any refractive surgery or cataract surgery, Rock inhibitors at dosesof 0.1 micrograms/ml to 40 micrograms/ml or more for topical applicationor Wnt inhibitor can be injected as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) in the corneal pocket or in theanterior chamber, or applied as drops in the post-operative period toreplace prednisolone or other steroids, or NASIDs, and encourageregrowth of the cut neurons in the cornea.

In one embodiment, after LASIK or any refractive surgery or cataractsurgery, Wnt inhibitors or, Rock inhibitors such as botulinum toxin(Botox) can be injected under the conjunctiva or applied as drops in thepost-operative period to encourage regrowth of the cut neurons in thecornea after LASIK or other corneal surgery at doses of 1 to 10 units ofBotox injected under the conjunctiva or 1-2 drops daily at concentrationof 10 picograms to 500 picograms of Botox in physiological solution ortopically as drops.

In one embodiment, in dry eye syndrome, Rock inhibitors or Wntinhibitor, such as botulinum toxin (Botox) can be applied as drops orinjected subconjunctivally to eliminate the inflammatory component ofdry eye at doses of 1-10 units once a month or once every 2 to 3 monthswith slow release nanoparticle conjugates in biodegradable polymers oras nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, in dry eye syndrome, Rock inhibitors such asbotulinum toxin (Botox), Fasudil, etc. or Wnt inhibitors, such asniclosamide, nitazoxanide (NTZ), ivermectin, FH535, IWP-2, PNU-74654,IWR-lendo. IWR-exo, Demethoxy Curcumin, sulforaphane as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) and vitamin Dcan be given orally at the tolerated dose or 40 mg Fasudil or 1 gramniclosamide or 10-100 units of Botox to eliminate the inflammatorycomponent of dry eye, sulforaphane at 400 micrograms and Vitamin D3000-5000 IU.

In one embodiment, the Rock inhibitors, such as Fasudil derivatives atconcentrations or 10 picograms to 10 nanograms to 1 microgram per dropBotox solution of 0.1 units of Botox can be administered with smallmolecule WNT inhibitors or a low concentration 1-10 micrograms.

In one embodiment, a topical or subconjunctival or intraocularadministration of the Rock inhibitors, such as Fasudil derivatives,etc., at concentrations or 10 picograms to 100 nanograms/0.25 ml orBotox solution of 0.1-1 units can be administered with small moleculeWNT inhibitors or a low concentration of sulforaphane and vitamin D toinhibit the inflammatory processes or auto-immune response as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, Rock inhibitors are administered with antibody coatednanoparticles conjugated with thermosensitive nanoparticles andAdalimumab, a humanized antibody administered topically orsubcutaneously at a non-toxic dose.

In one embodiment, Rock inhibitors are administered with antibody coatednanoparticles, dendrimers, liposomes, etc. to the conjunctiva asliposomes or ointment in Meibomian gland inflammation to releasemedication at a concentration of 1 picogram to 100 units or morepicograms/0.25 ml to 0.5 ml along with an antibiotic with nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, Wnt inhibitors or Rock inhibitors, such as Fasudilderivatives, etc. are administered with nanoparticles, dendrimers,thermosensitive polymers conjugated with polylactic or polyglycolic acidor chitosan, microspheres, liposomes, dendrimers, microparticles ornanoparticles, or porous silicone implant and combinations thereof, andthey are administered as drops, or injected in the conjunctival orlacrimal glands along with immunosuppressive agents, such asmycophenolic acid, etc. as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, topical administrations, subconjunctival injections,sub-tenon injections, suprachroideal injections, intravitreal injectionscan be combined with small molecule Wnt inhibitors or standardanti-inflammatory agents (e.g., steroids, dexamethasone, etc.) depot,nanoparticle implants, biodegradable or non-biodegradable polymers,NASIDs, Diclofenac, immunotherapy immunosuppressants, etc. to treatinflammatory processes of the lid conjunctiva or the cornea and the lidor throughout the day. For injection, a dose of about 50 picograms/ml toabout 200 micrograms/ml may be used as a surgical implant, for example,in a diffusible walled reservoir sutured to the wall of the sclera, ormay be contained within an inert carrier, such as microspheresdendrimers, or liposomes, porous silicon oxide microparticles or as nanoor microparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) to provide aslow-release drug delivery system.

In one embodiment, a formulation of Wnt or Rock inhibitors is used fromthe group consisting of topical administration at a concentration ofabout 50 picograms/ml to less than 1 micrograms/ml, subconjunctivalinjection at a dose in the range of about 1 picogram/ml to about 200micrograms/ml, intravitreal injection at a dose in the range of about0.1 picogram/ml to about 20 micrograms/ml, or retrobulbar injection at adose in the range of about 2 micrograms/ml to about 200 micrograms/ml inslow release microspheres or dendrimers. In one embodiment, aformulation of Wnt or Rock inhibitors is used comprising intraocularlyadministering to a patient after corneal surgery at picogram to nanogramconcentrations or as nano- or microparticles with (alpha)-cyclodextrin,or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) preparation.

In one embodiment, a formulation of Wnt or Rock inhibitors is used as acomposition consisting essentially of Rock inhibitors in apharmaceutically acceptable formulation and in an amount effective toenhance post-surgical to enhance ocular moisture, nerve regeneration inthe patient wherein the composition is administered at a concentrationsup to about 10 micrograms/ml by at least one of slow releasepolycaprolactone, polylactic, or polyglycolic acid, etc. over manymonths, intraocular administration of the composition or is administeredtopically at a concentration in the range between about 10 picograms/mlto less than 1 microgram/ml depending on the composition of themedication or as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, wherein the polymeric composition is administered bysubconjunctival injection at a dose in the range of about 1 picogram/mlto about 20 micrograms/ml, intravitreal injection at a dose in the rangeof about 1 picogram/0.1 ml to about 20 nanograms/ml, or retrobulbarinjection at a dose in the range of about 20 nanograms/ml to about 2micrograms/ml, as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, a formulation of Wnt or Rock inhibitors is used toenhance post-surgical ocular moisture or in papillitis, optic nerveneuritis, uveitis, or scleritis in the patient wherein the compositionis administered at a concentration up to about 50 picograms/ml by atleast one of intraocular injection, or the composition is administeredtopically at a concentration in the range between about 50 picograms/mlto less than 1 micrograms/ml as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, a formulation of Wnt or Rock inhibitors is usedwherein the composition is administered by subconjunctival injection ata dose in the range of about 1 picogram/ml to about 2 micrograms/ml,intravitreal injection at a dose in the range of about 1 nanogram/0.1 mlto about 20 nanograms/ml, or retrobulbar injection at a dose in therange of about 200 nanograms/ml to about 2 micrograms/ml as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, a method to treat an ocular condition in a patientcomprises intraocularly administering to the patient a pharmaceuticallyacceptable formulation of a drug selected from the group consisting ofRock inhibitors, such Rock inhibitors as Fasudil or derivatives innanogram to microgram concentrations in microspheres, dendrimers,physiological solution, botulinum toxin in picogram concentrations inpolymeric microspheres or 0.3-5 units injectable, or Wnt inhibitors,such as niclosamide, nitazoxanide (NTZ), ivermectin, nanogram tomicrogram concentration in microspheres, dendrimers, suspension oranother polymer, sulforaphane 10-400 nanograms in microspheres,dendrimers, or as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) or another polymer and Vitamin Dtaken orally in 1000-5000 IU etc., Fasudil derivatives taken orally 1-40mg, niclosamide orally in 10-500 mg tablets, sulforaphane in capsule10-40 mg or more ivermectin taken orally 1-400 mg or more and topicalformulation as drops, ointment, or gel in a non-toxic formulation forthe patient undergo surgery in the eye for refractive errors, diabeticretinopathy, retinal detachment, or after cataract surgery or refractivesurgery for the duration until the eye is free of inflammation and hasrecovered from the surgery.

In one embodiment, non-toxic doses of Rock inhibitors in an amount up toabout 1-200 micrograms/ml effective to treat dry eye or another ocularcondition selected from diabetic retinopathy, retinitis pigmentosa, orage related macular degeneration without substantial toxicity and atleast one Wnt inhibitor or Rock inhibitor, wherein the composition isadministered by at least one of intraocular injection at a concentrationup to about 2 picograms/ml, or the composition is administered topicallyat a concentration in the range between about 1 picograms/ml to lessthan 10 nanograms/ml as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, a formulation of Wnt or Rock inhibitors is used astopical administration at a concentration between about 50 picograms/mlto 200 nanograms/ml, subconjunctival injection at a dose in the range ofabout 1 picograms/ml to about 20 micrograms/ml in slow release polymer,intravitreal injection at a dose in the range of about 1 picogram/0.1 mlto about 2 micrograms/ml, or retrobulbar injection at a dose in therange of about 1 picograms/ml to about 200 nanograms/ml suspension inslow release polymer depending on the composition of the medication asnano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) preparation.

In one embodiment, a method to treat an ocular condition in a patient byintraocularly administering a pharmaceutically acceptable formulation ofWnt inhibitors or rock inhibitors in an amount effective to treat thecondition. The method provides treatment while avoiding systemicadministration of systemic medication. In one embodiment, a sustainedrelease pharmaceutically acceptable formulation is implantedintraocularly in polymeric slow release compound having about 20nanograms to 1 microgram or more of Fasudil to about 0.1 micrograms to40 micrograms or 1 milligram of Fasudil or other rock inhibitorsimplanted in or on the eye and may continuously deliver Fasudil for fiveor more years.

In another embodiment, a concentration up to about 10 or more microgramsof Rock inhibitors is administered intraocularly without substantialtoxicity.

In another embodiment. Fasudil derivatives are taken orally 1-40 mg,niclosamide is taken orally in 10-500 mg tablets, sulforaphane is takenorally in capsule 10-40 mg or more, ivermectin is taken orally 1-400 mgor more, and topical formulations may be administered as drops,ointment, gel in a non-toxic formulation.

In another embodiment, Rock inhibitors at a concentration in the rangeof about 1 picogram/ml (0.0000000001%) to less than 0.1 micrograms/ml(less than 0.001%) is administered topically. In other embodiments,Fasudil or another Rock inhibitor at a concentration in the range ofabout 1 nanogram/ml to about 200 micrograms/ml is injected under theconjunctiva, or a concentration in the range of about 1 picogram/0.1 mlto about 200 micrograms/ml is injected in the vitreous, or aconcentration in the range of about 20 picograms/ml to about 200nanograms/ml is injected behind the eyeball as nano- or microparticleswith (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) preparation.

In one embodiment, the Rock inhibitors, such as Fasudil, etc., or Wntinhibitors, such as niclosamide, are administered as topical or a sprayat non-toxic concentrations of 1 picogram/ml to 20 nanograms/ml in aphysiological pH balanced, with osmolarity of 310 to prevent and treat,decrease the time of onset, or lessen the severity of a wide variety ofdiseases such as lichen planus, ocular conditions, such as retinitispigmentosa, ocular irritation following corneal surgery (e.g., LASIKsurgery), age related macular degeneration, diabetic retinopathy, dryeye disease, scleritis, papillitis, and uveitis, scleritis parsplanatis, or vogt-koyanagii syndrome.

In one embodiment, the Rock inhibitors, such as Fasudil, etc., or Wntinhibitors, such as niclosamide, nitazoxanide (NTZ), are administered astopical or a spray at non-toxic concentrations of 1 picogram/ml to 20nanograms/m or in an ointment or cream or suspension of microspheres anddendrimers in meibomian gland disease as nano or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) preparation.

In one embodiment, the Rock inhibitors, such as Botulinum toxins areadministered as topical or a spray at non-toxic concentrations of 1picogram/ml to 1 nanograms in a cream, ointment, suspension ofmicrospheres or dendrimers, etc. for topical application in lichenplanus, nerve damage after LASIK or refractive surgery procedures, ordiabetes or wrinkle treatment.

In one embodiment, the Rock inhibitors, such as Fasudil 40-80 mg/kg,etc., or Wnt inhibitors, such as niclosamide, 100-500 mg or ivermectin,250 mg to 2000 mg are administered orally to prevent and treat, decreasethe time of onset, or lessen the severity of a wide variety of diseases,such as optic nerve neuritis, papillitis, variety of idiopathic uveitis,scleritis, or ocular conditions, such as retinitis pigmentosa, ocularirritation following corneal surgery (e.g., LASIK surgery), age relatedmacular degeneration, diabetic retinopathy, dry eye disease, papillitis,or uveitis.

In one embodiment, the Rock inhibitors or Wnt inhibitors areadministered as topical or a spray at non-toxic concentrations of 1picogram/ml to 20 nanograms/ml in a physiological pH balanced solutionwith osmolality of 310 to treat the corneal nerve cuts after LASIKsurgery to decrease inflammatory process and encourage fast regrowth ofneurons from the cut end of the corneal nerves and enhance cornealsensation recovery time and prevent dry eye formation.

In one embodiment, the Rock inhibitors (40-80 mg/Kg) or Wnt inhibitorsare administered orally after LASIK surgery to decrease inflammatoryprocess and to encourage fast regrowth of neurons from the cut end ofthe corneal nerves and enhance corneal sensation recovery time andprevent dry eye formation.

Another embodiment of the invention is a method to treat ocularconditions including ocular irritation following corneal surgery,conjunctivitis, canaliculitis or schlemm's canal of the eye, iritis,lacrimal and Meibomian glandes are treated with Rock inhibitors, such asFasudil or its derivatives in nanogram to microgram concentrations inmicrospheres, dendrimers, physiological solution, Botulinum toxin inpicogram concentrations in polymeric microspheres dendrimers, or 0.3-5unit injectable, or Wnt inhibitors, such as niclosamide, nitazoxanide(NTZ), ivermectin, nanogram to microgram concentration in microspheressuspension or another polymer, sulforaphane 10-400 nanogram inmicrospheres, dendrimers, or another polymer and Vitamin D taken orallyin 1000-5000 IU, etc.

In one embodiment, a sustained release pharmaceutically acceptableformulation is implanted intraocularly. For example, a matrix containingin the range of between about 0.4 to 1 mg Fasudil can last for ten ormore years. In another embodiment, a concentration up to about 1microgram Fasudil or others Rock inhibitors is administeredintraocularly, inside the joint in arthritis, or subcutaneously orsub-gingival injection in lichen planus without substantial toxicity asnano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin, orhydroxypropyl-b-cyclodextrin (bHPCD).

In another embodiment, Rock inhibitors at a concentration in the rangeof about 1 nanogram/ml (0.0000001%) to less than 1 microgram/ml (lessthan 0.0001%) are administered topically. In other embodiments, Fasudilat a concentration in the range of about 1 nanogram/ml to about 20microgram/ml is injected under the conjunctiva, or a concentration inthe range of about 1 nanogram/0.1 ml to about 200 micrograms/ml isinjected in the vitreous, or a concentration in the range of about 20nanograms/ml to about 20 micrograms/ml is injected in a slow releasepolymer, such as polycaprolactone or polylactic or glycolic, in thevitreous cavity or behind the eyeball or other part of the body asneeded.

In another embodiment, Rock inhibitors at a concentration in the rangeof about 1 nanogram/ml (0.0000001%) to less than 1 microgram/ml (lessthan 0.0001%) are administered topically. In other embodiments, Fasudilat a concentration in the range of about 1 nanogram/ml to about 20micrograms/ml is injected under the conjunctiva, or a concentration inthe range of about 1 nanogram/0.1 ml to about 200 microgram/ml isinjected in the vitreous, or a concentration in the range of about 20nanogram/ml to about 20 micrograms/ml is injected in a slow releasepolymer, such as polycaprolactone or polylactic or glycolic, in thevitreous cavity or behind the eyeball in subconjunctival space, orsubcutaneously as needed as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) preparation.

In one embodiment, the non-toxic doses of Wnt inhibitors, Rockinhibitors, or Botox, act as an anti-inflammatory agent. The botulinumtoxin or botox preparation may be administered topically to the eye oreye lid, forehead skin at 1 pictogram to 1 nanogram concentrations, 1pictogram to 5 nanogram concentrations, for example, using drops, anointment, a cream, a gel, a suspension of microsphere, dendrimers, etc.The agent(s) may be formulated with excipients such as methylcellulose,hydroxypropyl methylcellulose, hydroxypropyl cellulose, the LD50s of anynaturally occurring botulinum toxin protein is at 1.3 nanograms perkilogram (abbreviated ng/kg). In a 75 kg (165 lbs.) subjects, the LD50for botulinum toxin would be 97.5 nanograms if injected directly into avein or artery. 100 unit vials contains 0.75 nanograms=750 picograms ofbotulinum toxin A in the entire vial.

In one embodiment, a dose of botulinum toxin in 100-2000 picograms willnot be toxic if injected subcutaneously, or 750 picograms (100 units)1-2 times a month will not be toxic. Higher doses can be used withcaution and it would be desirable not to exceed these levels to preventan immune response to the medication.

In one embodiment, a dose of botulinum toxin in 100-2000 picograms willnot be toxic if injected subcutaneously, or 750 picograms (100 units)1-2 times a month will not be toxic. Higher doses can be used withcaution and it would be desirable not to exceed these levels to preventan immune response to the medication.

In one embodiment, the concentrations 1-20 picograms/Botox in aphysiological solution, or up to 30 picograms conjugated with antibodycoated nanoparticles would be non-toxic to the body or when conjugatedwith thermosensitive polymeric coating of the nanoparticles in aphysiologic solution or used as drops or injectable or as nano- ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) preparation.

In one embodiment, the Wnt inhibitors or Rock inhibitors may be injectedinto the eye, for example, injection under the conjunctiva or tenoncapsule, intravitreal injection, or retrobulbar injection as a slowrelease nanoparticle. The agent(s) may be administered with a slowrelease drug delivery system, such as polymers, matrices, microcapsules,nanoparticles or microparticles of porous silicone or other deliverysystems formulated from, for example, glycolic acid, lactic acid,combinations of glycolic and lactic acid, liposomes, silicone, as nano-or microparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) orpolyanhydride polyvinyl acetate alone or in combination withpolyethylene glycol, etc. The delivery device can be implantedintraocularly, in the lens capsule, over the lens in the choroid forexample, implanted under the conjunctiva, implanted in the wall of theeye, sutured to the sclera, for long-term drug delivery or injected inthe vitreous cavity.

In one embodiment, one uses a composition containing Rock inhibitors,such as Fasudil etc., at a concentration in the range of about 50picogram/ml (0.000000005%) to about 50 micrograms/ml (0.005%),niclosamide at a concentration in the range of about 50 picograms/ml toabout 50 micrograms/ml, or a combination of Fasudil or an immunesuppressive agent, such as mycophenolic acid, to achieve a totalconcentration of both agents of about 50 picogram/ml to about 50microgram/ml as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD). Within this range, the agent(s)has wide safety and efficacy, permitting specific doses oradministration protocols to be formulated for specific applications. Forexample, some patients may prefer once a day administration compared toadministration more than once a day, so a higher concentration ofagent(s) may be used for these patients.

In another embodiment, Rock inhibitors, such as Fasudil may also beadministered by injection. Intraocular injection may be desirable ornecessary, for example, for conditions in which topical administrationis either not advised or is inadequate, for patients who have difficultyself-administering medications, etc. In one embodiment, the volumeinjected is less than 0.3 ml. In another embodiment, the volume injectedis in the range of about 0.01 ml to about 0.3 ml. For intravitrealadministration (injection into the vitreous), Rock inhibitorconcentrations in the range of about 1 nanogram/0.1 ml to about 20microgram/ml (0.002%) may be used without toxicity or adverse sideeffects.

In another embodiment, niclosamide used in amounts ranging from about 1nanogram to about 10 micrograms is contained in an aqueous-based creamexcipient. In another embodiment, the amount of Fasudil, etc. or otherRock inhibitors ranges from about 1 nanogram to about 10 micrograms, andis contained in an aqueous-based cream excipient. In another embodiment,Fasudil and niclosamide or mycophenolic acid are present in anaqueous-based cream excipient in various proportions. In anotherembodiment, to achieve a total amount of combined agents of about 1nanogram to about 10 micrograms, the drug(s) may be incorporateddirectly into the cream in solution, or may be contained in liposomes ormicrospheres, dendrimers, either in solution or in an anhydrous form asnano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD). The cream formulation is usuallyapplied to the eye at bedtime, but it may be applied any time throughoutthe day if the cream does not cause blurred vision. In anotherembodiment, the agent(s) is formulated as a solution or suspension andis applied topically in the form of eye drops.

In another embodiment, for long term delivery of a Rock inhibitor or aWnt inhibitor, either alone or in combination, and/or for sustainedrelease, a matrix housing or containing the agent(s) may be implantedinto the eye. For example, a reservoir containing in the range of about1 milligram to about 5 milligrams of agent(s) is estimated to be able torelease about 1 microgram agent(s) per day. At such a release rate,continuous, sustained dosing may occur over 1000 to 5000 days. If lessthan 1 microgram of agent(s) per day is released, sustained dosing maylast up to or more than a decade. In one embodiment, less than 50micrograms/day of agent(s) is released from the matrix. In anotherembodiment, agent(s) is released form the matrix at a rate in the rangeof about 50 picogram/day to about 50 micrograms/day. In anotherembodiment, agent(s) is released from the matrix at a rate in the rangeof about 1 micrograms/day to about 5 micrograms/day.

In another embodiment, a surgically implanted intraocular device ormatrix may be provided with a reservoir container having a diffusiblewall of polyvinyl alcohol or polyvinyl acetate or polycprolactone andcontaining milligram quantities of a Rock inhibitor or Wnt inhibitor, ora combination of them may be implanted in the sclera. As anotherexample, milligram quantities of agent(s) may be incorporated into apolymeric matrix having dimensions of about 1 millimeter (mm) by 2millimeter (mm), and made of a polymer such as polycaprolactone,poly(glycolic) acid, poly(lactic) acid, or a polyanhydride, or a lipidsuch as sebacic acid, and may be implanted on the sclera or in the eye.

In another embodiment, as one example of an inert matrix, liposomes maybe prepared from dipalmitoyl phosphatidylcholine (DPPC), preferablyprepared from egg phosphatidylcholine (PC) since this lipid has a lowheat transition. Liposomes are made using standard procedures as knownto one skilled in the art. The agent(s), in amounts ranging frompicogram to microgram quantities, is added to a solution of egg PC, andthe lipophilic drug binds to the liposome.

In another embodiment, the implantable formation may be in the form of acapsule of any of the polymers previously disclosed (e.g.,polycaprolactone, poly(glycolic) acid, poly(lactic) acid, polyanhydride)or lipids that may be formulated as microspheres or dendrimers. As anillustrative example, Fasudil may be mixed with polyvinyl alcohol (PVA),the mixture then dried and coated with ethylene vinyl acetate, thencooled again with PVA. Niclosamide bound with liposomes may be appliedtopically, either in the form of drops or as an aqueous based cream, ormay be injected intraocularly as nano or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD). In a formulation for topicalapplication, the drug is slowly released over time as the liposomecapsule degrades due to wear and tear from the eye surface. In aformulation for intraocular injection, the liposome capsule degrades dueto cellular digestion, other slow release polymers such as PLA, PGA,Polycaprolactone, microsphere, dendrimers) or as nano- or microparticleswith (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) are alsoutilized.

In another embodiment, the time-release administration, however, isformulated so that the concentration released at any period of time doesnot exceed a toxic amount. This is accomplished, for example, throughvarious formulations of the vehicle (coated or uncoated microspheres,coated or uncoated capsule, lipids, dendrimers, or polymer components,unilamellar or multilamellar structure, and combinations of the above,etc.). Other variables may include the patient'spharmacokinetic-pharmacodynamic parameters (e.g., body mass, gender,plasma clearance rate, hepatic function, etc.). The formation andloading of microspheres, dendrimers, microcapsules, liposomes, etc. andtheir ocular implantation are standard techniques known by one skilledin the art.

In one embodiment, Rock inhibitors, such as Fasudil, or Botox, etc. orWnt inhibitors such as niclosamide, alone or in combination with lowmolecular weight heparin and metalloproteinase inhibitors, such asdoxycycline, tetracycline, etc. can be used at non-toxic concentrationswith or without dexamethasone, for dry eye or lichen planus or pemphigusor Stevens-Johnson syndrome, lesions of the mucosa, or skin or otherinflammatory diseases of the retina, cornea, conjunctival sclera oroptic nerve neuritis, scleritis, uveitis in an appropriate physiologicalsolution or ointment, as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD), etc.

In one embodiment, the intravenous solution form of Rock inhibitors orWnt inhibitors may be diluted to achieve the indicated concentrationusing 0.9% NaCl or 5% dextrose, or an organic solvent such as dimethylsulfoxide (DMSO) or sterol, lanosterol, squalene, and/or squalamine.Intraocular administration may be any of the routes and formulationspreviously described. For injection, either a solution, emulsion,suspension of a liquid, capsular formulation of microspheres,dendrimers, or liposomes, as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD), etc. may be used.

In one embodiment, Rock inhibitors or Wnt inhibitors or Botox may beinjected subconjunctivally to treat uveitis at a dose in the range ofabout 1 picogram/ml to about 200 picograms/ml, or intravitreally at adose of about 1 gram/0.1 ml to about 200 picograms/ml. In oneembodiment, the dose is about 50 picograms/0.1 ml. To treat scleritisinvolving the anterior sclera, Rock inhibitors or Wnt inhibitors orBotox may be administered topically as nano- or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,or hydroxypropyl-b-cyclodextrin (bHPCD).

In one embodiment, Rock inhibitors or Wnt inhibitors or Botox may beinjected to treat scleritis involving the posterior sclera, may beadministered by retrobulbar injection at a dose in the range of about 20picogram/ml to about 800 picograms/ml or more and dissolved in DMSO or avery low concentration of alcohol or sterol, lanosterol, squalene,and/or squalamine.

In one embodiment, to treat neuritis or papillitis, Rock inhibitors maybe administered by retrobulbar injection at a dose in the range of about200 picogram/ml to about 800 nanograms/ml of Fasudil and itsderivatives, etc.

In one embodiment, to treat neuritis or papillitis, Rock inhibitors(e.g., Fasudil) may be administered orally at a dose in the range ofabout 40-80 milligrams of Fasudil tablets, etc. or one time niclosamide1-2 grams orally.

In one embodiment, the ocular solutions contain at least one Rockinhibitor or Wnt inhibitor such as sulforaphane and provideanti-inflammatory, anti-cell proliferation, anti-cell migration effectsif given orally with Vitamin D, topically as dendrimer or microspheredelivery or an injectable non-toxic preparation.

In one embodiment, the solution or suspension is administeredintraocularly after cataract surgery before insertion of a replacementintraocular lens, resulting in reduced post-operative inflammation,which may eliminate the need for a steroid therapy.

In one embodiment, the solution may be one that is invasivelyadministered, for example, an irrigation or volume replacement solutioncontaining at least one Rock inhibitor, such as Botox, or Wnt inhibitor.

In one embodiment, the solution may be one that is non-invasively ortopically administered in the form of drops, ointments, gels, creams,etc. and may include eye lubricants and contact lens solutions. Thesolution may contain a supratherapeutic concentration of agent(s), suchas 40 microgram/ml or to 80 micrograms/ml or more for topicalapplication ranges 40 nanograms/ml to 4 micrograms/ml Fasudil and itsderivatives, etc. as nano- or microparticles with (alpha)-cyclodextrin,or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) so that a therapeutic concentrationof a topically administered solution accumulates in a diseased ocularstructure sufficient to treat the disease.

In one embodiment, medications are administered with antibody coatednanoparticles, dendrimers, or as nano or microparticles with(alpha)-cyclodextrin, or (beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD), thermosensitive polymers,nanoparticles, dendrimers, lactic or glycolic acid, chitosan orcombinations, etc. Immunosuppressives are all conjugated with theantibody coated nanoparticles for slow release as topical drops or aninjectable preparation for dry eye after LASIK, meibomian glandinflammation, optic nerve neuritis, uveitis, scleritis, etc.

In one embodiment, Rock inhibitor or Wnt inhibitors are administered bytopical drops, spray, subconjunctival injection, subtenon injection,suprachroideal injection, intravitreal injection in combination withstandard anti-inflammatory agents etc. and steroids, dexamethasone, etc.as depot, nanoparticles implant biodegradable or non-biodegradablepolymers.

In one embodiment, a method of using Rock inhibitors or Wnt inhibitorsis disclosed where Rock inhibitors or Wnt inhibitors are administered atnon-toxic doses to the skin or mucosa.

In one embodiment, in treating lichen planus, for example, a topicaladministration may contain between about 10 picogram/ml drug to about 50micrograms/ml of Fasudil, etc. or other Rock inhibitors in a formulationwhich may be applied at bedtime or throughout the day or as aninjection, a dose of about 50 picograms/ml to about 200 micrograms/mlaround or inside the lesion. In one embodiment, the medication may beused as a surgical implant, for example, in a diffusible walledreservoir sutured to the surrounding tissue, or may be contained withinan inert carrier, such as microspheres, dendrimers, or liposomes,microparticles or nanoparticles, or porous silicone implant to provide aslow-release drug delivery system to release the medication at 1picogram to 100 picograms (e.g., Fasudil, etc.) per day.

In one embodiment, a formulation of Wnt or Rock inhibitors is used totreat ocular conditions, such as dry eye disease, as well as otherconditions, is disclosed. Rock inhibitors and Wnt inhibitors are used ass topical drop spray application or injection into the eye, orimplantation in or on the eye. For example, a topical administration maycontain between about 10 picograms/ml drug to about 50 micrograms/mldrug in a formulation which may be applied at bedtime.

In one embodiment, the patient is administered with Rock inhibitors orWnt inhibitors as nano- or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD), microparticles or nanoparticles,or porous silicone implant alone or in combination with NSAIDs or totreat associated chronic pain, with more or less inflammatory processesas seen in the eye after refractive surgery or vitreoretinal surgery orocular hypotony caused either by surgery and associated systemic diseasesuch as diabetes or autoimmune uveitis.

In one embodiment, non-toxic doses of Rock inhibitors, such as Fasudil,etc., 200 picograms to 2 nanograms or as Botox (10-100 units) areadministered locally at multiple locations in treatment or pain orchronic inflammation.

In one embodiment, an intrastromal corneal implant is prepared forimplantation inside a patient's corneal stroma, and is comprised of anorganic stroma, a synthetic 3-D printed corneal stroma, a hybridpolymeric/organic stroma, a genetically modified animal cornea stroma,an eye bank corneal stroma, or tissue culture grown corneal stroma,which are made non-immunogenic to the host cornea by cross-linking theintrastromal corneal implant with riboflavin solution and UV radiation.

In one embodiment, after the enucleated eyes are obtained from thecornea bank, the eyes are dipped in povidone-iodine to sterilize thesurface of the tissue, rinsed with saline solution, and then, using aQ-tipped applicator dipped in a saline solution. The corneal epitheliumis removed from the donor cornea, then a standard 6-7 mm circulartrephine cuts though the remaining cornea and a circular remainingcorneal tissue is removed from the eye, cleansed again and theendothelial cells of the cornea are removed while leaving the cornealstromal with its cells.

In one embodiment, using a femtosecond laser or microkeratome, acircular piece of the donor corneal stroma is cut with the thickness of50, 100, or 150 microns or more thickness that serves for correcting therefractive error of the human recipient cornea from <+1.00 D to +15 D or<−1.00 D to −15.00 power and up to ±6.00 D power of astigmatismincluding lower and higher order of aberrations.

In one embodiment, the donor stromal inlay is sterilized by dipping itin a solution of 0.1-5% photosensitizer or riboflavin, or riboflavinnanoparticles conjugated with cell penetrating peptides (CPP) oractivatable cell penetrating peptides (ACPP) to penetrate the entirestromal block. In one embodiment, the riboflavin solution contains othermedications such as NSAIDS, Rock inhibitors, Wnt inhibitors, integrininhibitors or GSK inhibitors in a polymeric slow release polylactic,glycolic, polycaprolactone, micelles, porous silicon, etc. to releasefor 3-4 weeks or more. In one embodiment, the riboflavin has theosmolarity of about 300 mOsmol, pH of about 7, with or without dextranor low molecular heparin or hyaluronic acid, and polymericnanoparticles.

In one embodiment, the corneal inlay containing riboflavin is irradiatedwith the UV light of a laser with the wavelength of 350-390 nm with apower of 3-30 milliW/cm2 for a period of 1-10 minutes or more tocross-link the inlay by a combination effect of riboflavin and UVradiation creating singlet oxygen and reactive species that cross-linkproteins making the inlay non-immunogenic regardless of its origin, suchas human or animal of genetically modified animals, such as pig fororgan transplant, etc., and the cross-linking simultaneously sterilizesthe implant by killing all the potentially existing pathogens.

In one embodiment, the implant is made more permeable to a riboflavinsolution by applying femtosecond laser pulses to it (see FIGS. 54A and54B), then cross-linking by a combination effect of riboflavin and UVradiation (see FIG. 55) creating singlet oxygen and reactive speciesthat cross-link proteins making the inlay non-immunogenic andsimultaneously sterilizing the implant by killing all the potentiallyexisting pathogens. The femtosecond laser will create some air bubblesinitially, which rapidly dissipate in the air, and do not change thethickness of the inlay. As shown in FIGS. 54A and 54B, small cavities orholes may be formed in the corneal inlay or implant 810 using afemtosecond laser (i.e., the small cavities or holes are formed in thecorneal inlay or implant 810 using the laser beam(s) 812 emitted fromthe femtosecond laser) so as to make the corneal inlay or implant 810more permeable to a photosensitizer solution (e.g., riboflavinsolution), thereby allowing more of the photosensitizer solution to getinside the corneal inlay or implant 810. In the illustrative embodiment,the corneal inlay or implant 810 of FIGS. 54A and 54B is formed from adonor human cornea, and has an outside diameter between 6 and 7millimeters. As shown in FIGS. 55, after the corneal inlay or implant810 has been soaked in the photosensitizer solution (e.g., theriboflavin solution), the corneal inlay or implant 810 is irradiatedusing ultraviolet (UV) radiation 814 so as to cross-link the cornealinlay or implant 810. The photosensitizer solution (e.g., the riboflavinsolution) in which the corneal inlay or implant 810 is soaked may alsocontain one or more medications for treating the recipient eye of thepatient (e.g., one or more medications described above, such as Rockinhibitors, Wnt inhibitors, integrin inhibitors, GSK inhibitors, etc.).

In another embodiment, the donor cornea, genetically modified cornea, orhumanized cornea is prepared by removing it from the donor eye using a 6to 8 millimeter diameter trephine and scissors to separate it from theeye. The corneal epithelium and endothelium is removed chemically,mechanically, or combination thereof using a Q-tipped applicator dippedeither in saline or a solution of 1% or more in water or saline solutionand subsequently rinsed immediately with saline to remove the remainingalcohol and/or the loose cells to create a corneal stromal tissue byusing a standard femtosecond laser or a mechanical microkeratome tocreate two or three stromal implant circles of about 100-150 microns inthickness (e.g., see FIG. 61A), when exposed to a solution of 1-5%riboflavin nanoparticles and cell penetrating peptides (CPP), and ifneeded, an appropriate medication and/or another photosensitizer tocross-link the implant rapidly in less than 3 minutes. Then, the implantis radiated using either a laser with UV radiation of wavelength ofbetween 380 nm to 390 nm, or other wavelength when anotherphotosensitizer rather than riboflavin is used, thereby crosslinking thecorneal proteins, including collagen, elastin, etc., and stiffening thecorneal collagen and simultaneously killing corneal stromal cells andall pathogens present in the implant and making the implantnon-immunogenic without affecting its transparency.

In another embodiment, a 3-D printed cornea is prepared without havingthe epithelial cells or the endothelial cells, or having only thestromal fibrils, and other proteins, such as collagen and elastin, etc.and mesenchymal stem cells to create a corneal stromal tissue, and byusing a standard femtosecond laser or a mechanical microkeratome, createtwo or three stromal implant circles of about 100-150 microns inthickness, exposed to a solution of 1-5% riboflavin or riboflavinnanoparticles and cell penetrating peptides (CPP) or anotherphotosensitizer to cross-link the implant rapidly in less than 3minutes. Then, the implant is radiated using a laser with UV radiationof wavelength of 380 nm to 390 nm, a power or 3 milliW/cm2 to 10milliW/cm2 or more for a time of 1-10 minutes as needed forcross-linking the corneal proteins, including collagen, elastin, etc. tostiffen the corneal collagen and simultaneously kill corneal stromalcells and all pathogens if present in the implant, and make the implantnon-immunogenic by crosslinking it, without affecting its transparency.

In one embodiment, the implant is modified to be able to pass through asmall incision so as to be placed with an instrument under a flap of thecornea, such as after a LASIK procedure or a procedure that does notrequire a large corneal incision cutting more corneal nerves. Byinserting the implant through a small incision into a stromal pocket,the potential side effects of corneal anesthesia and dry eye areprevented, such as in the small incision lenticule extraction (SMILE)procedure. However in the SMILE procedure, the removed stromal lenticulerequires scratching the bordering stroma to the explant, the previouslycut stromal lenticule with femtosecond laser inside the stroma forcorrection of low myopia. However in this typical SMILE procedure, theremoved tissue (stromal lenticule) is not replaced with an appropriatelenticule, which would provide a smooth border and shape that reduces orprevents the post-operative glare experienced by majority of thepatients.

In one embodiment, in the SMILE procedure, the stromal pocket is notleft empty, but is filled with a small porous human corneal,cross-linked implant with an appropriate size and shape to achieve therequired refractive correction, while preventing the post-operativeglare in this patient.

In one embodiment, the surrounding tissue of the inlay is alsocross-linked when the implant has some crosslinker in it, wherein thecross-linker diffuses out of the implant and penetrates the surroundingtissue when the eye is exposed to an external UV radiation for a shortperiod of time after implantation.

In one embodiment, the inlay is placed after a LASIK flap is prepared,and the refractive error of the eye regardless of being myopic,hyperopic, astigmatic, or having any other combined refractive errors,is corrected using a standard excimer laser equipped with wavefronttechnology and Shack-Hartmann wavefront sensor during in vivo surgeryobjectively and corrected with the excimer laser immediately, then thecorneal flap is repositioned, and the appropriate antibiotic and othermedications are applied to the eye.

In one or more embodiments, referring to FIGS. 56-59, a LASIK flap 820is formed in a cornea 818 of an eye 816 using a femtosecond laser cut822 (see FIG. 57). Then, a corneal inlay or implant 824 is insertedunder the flap 820. The inlay or implant 824 is cross-linked so as toprevent an immune response to the implant and/or rejection of theimplant 824 by the patient. Then, as shown in FIG. 58, laser energy 826is applied to the implant 824 in the eye using an excimer laser so as tomodify the refractive power of the implant 824 while being monitoredusing a Shack-Hartmann wavefront system so as to achieve a desiredrefractive power for the implant. Finally, as shown in FIG. 59 thecross-linked implant 824 is covered with the flap 820, the cross-linkedimplant being surrounded entirely by the stromal tissue of the cornea.

In one embodiment, the stromal inlay is a single piece and does not haveany curvature to correct the refractive error and as such it is“refractively” neutral with an index of refraction of the normal corneaof 1.33 which similar to the water inside the stroma, and the inlay doesnot change the refractive power of the cornea, but can increase thethickness of the cornea. In one embodiment, a larger diameter inlay of 8to 12 mm in diameter and a thickness of 50 to 150 microns can be used ina patient with keratoconus to prevent corneal ectasia or in a patientwho has ectasia after refractive surgery, or in combination withphoto-therapeutic keratectomy, or as LASIK where the inlay is ablatedwith an excimer laser to correct the refractive power of the eye. Thiscould be a one step or two step procedure after the initial healing hastaken place.

In one embodiment, the implant is used in two different procedures. Inone embodiment, in a LASIK procedure, the implant is positioned over theexposed corneal stromal surface after creating a cornea stromal flap,and after ablation the inlay corrects the refractive error of the eye byusing an excimer laser and wavefront technology that controls the degreeand shape of the tissue removed from the inlay during the surgery toeliminate refractive errors of each eye individually during the surgery,then the corneal stromal flap is returned back over the reshapedanterior surface of the inlay and ultimately the reshaped inlay issurrounded on all sides by the corneal stroma and away from the host'sBowman's membrane or corneal epithelium.

In another embodiment, a small incision 852 (e.g., an incision havinglength of between 4 and 5 millimeters) is created in the cornea 850 ofthe eye 848 for inlay implantation inside the cornea 850. Initially a3-D cavity is created inside the corneal stroma with a femtosecondlaser, and through the small incision 852 the folded pieces of the inlay830, 832 or 836, 838, or preferably a composite lens, are implantedinside the stromal cavity as shown in FIGS. 62 and 63. A sidecross-sectional view of the stromal cavity 854 of the cornea 850 isshown in FIG. 64, while an implant or inlay 856 is shown disposed in thecavity 854 of the cornea 850 in FIG. 65.

A first exemplary composite inlay or implant 828 is illustrated in FIG.61A. The composite two-piece inlay or implant 828 in FIG. 61A comprisesa central portion 830 and a peripheral portion 832. A second exemplarycomposite inlay or implant 834 is illustrated in FIG. 61B. The compositetwo-piece inlay or implant 834 in FIG. 61B is transversely divided intotwo pieces, and comprises a first section 836 and a second section 838.A third exemplary composite inlay or implant 840 is illustrated in FIG.61C. The composite three-piece inlay or implant 840 in FIG. 61C istransversely divided into three pieces, and comprises a first section842, a second section 844, and a third section 846.

In one embodiment, the implant or inlay is reshaped prior toimplantation inside a 3-D pocket made with a femtosecond laser insidethe corneal stroma, with or without the removing of the stromal tissue,e.g., after removal of a part of the scarred stromal tissue or a stromallenticule, as performed in known SMILE procedure, to fill the cavitywith an implant which has a smooth surface and appropriate refractionmatching the needed refraction of the eye, using an excimer laser toablate the surface of the inlay. In one embodiment, the implant or inlayis folded and implanted inside the corneal stromal cavity. In anotherembodiment, after correcting the inlay to compensate for the refractiveerror of the patient's eye by using an excimer laser and wavefronttechnology, the inlay is cut into various pieces and shapes, thuspermitting the pieces of the inlay to be implanted inside the stromalcavity through a small incision (e.g., an incision having length ofbetween 4 and 5 millimeters), and simultaneously eliminating the oftenseen significant glare after the SMILE procedure that lasts about onemonth or more. The SMILE surgery involves cutting a small 3-D lenticuleinside the corneal stroma with a femtosecond laser, then digging outthis lenticule with the help of a bent-tipped needle shaped like a hook,but because the femtosecond laser does not completely separates thelenticule from the rest of the stroma, many attempts are done toseparate the lenticule from the stroma in a space where there is almostno room for manipulation, thus many scratches are created in the wall ofthe cavity, which is left to itself to heal. These surfaceirregularities with bits of the pieces of the left over stromal tissuerefract the incoming light and produce glare in extreme situations likescratched on or inside the reading glasses, etc. However, after a monthor more, gradually the tissue heals and the glare is reduced oreliminated.

In one embodiment, the post-operative glare can be significantly reducedby implanting a thin non-refracting one-piece inlay or a composite inlayinside the cavity left in the stroma after SMILE procedure where theinlay has a very smooth surface.

In one embodiment, after the SMILE procedure, the stromal cavity can beinjected with riboflavin and the excess solution removed immediately byexerting a minor pressure on the cornea to empty the cavity through thesmall incision in the cornea, the cornea is radiated for about 1-5minutes with UV radiation to cross-link the wall of the cavity. In oneembodiment, after the SMILE procedure, a cross-linker is mixed with avery dilute collagen, or elastin, or just a saline solution to wash outthe cavity and immediately removed under mild pressure to cross-link thearea so that the stroma with UV radiation heals and strengthens thecorneal resiliency despite removal of a part of the corneal stroma.

In another embodiment, composite lenses of implants are used to simplifyinsertion of the implant through a small incision 852 (see FIGS. 62 and63) using forceps with the folded pieces of the inlay and a lubricatingagent, such as low molecular weight heparin or hyaluronic acid, and thenopen the inlay with a tiny plastic rod inside the corneal cavity.

In one embodiment, the composite lenses are inserted in the stromalcavity with ease using each piece separately then joining the piecesinside the stromal cavity with or without lubricating agents. Becausethese lenses are organic and/or formed from a human corneal stroma, theyjoin easily together and the smooth cut edges heal easily withoutproducing significant glare in the post-operative period.

In another embodiment, the implant is a hybrid lens made from partiallyhuman cross-linked corneal tissue supported by a more resilientpolymeric coating such as polyethylene glycol (PEG), chitosan, oranother polymer etc. that is water permeable while providing moreresiliency to the implant and keeps the inlay surfaces smooth and easyto implant them through a small incision.

In another embodiment, a ribbed, perforated, or sieved polymericstructure (see FIGS. 66-69C) provides support to the overlaying humancross-linked cornea, which drapes over the polymeric structure duringthe implantation thereof, while the polymeric structure does not preventthe free flow of the fluid from the back of the cornea to the anteriorpart of the cornea and the holes in this polymeric structure are filledby the surrounding tissue and mostly the fluid in the cornea. Initially,with collective reference to FIGS. 66-68, the cross-linked donor cornealimplant or inlay 858 depicted in FIG. 66 may be placed on the polymericinlay back support structure 860 of FIG. 67 to form the hybrid lensimplant depicted in FIG. 68. As shown in FIGS. 67 and 68, the polymericinlay back support structure 860 is provided with a plurality of tinyholes 862 (e.g., having a diameter of 20 microns to 1 millimeter)disposed therethrough to enable free flow of the fluid from the back ofthe cornea to the anterior part of the cornea and the holes in thispolymeric structure. In the illustrative embodiment, the organiccross-linked corneal implant 858 (e.g., formed from donor cornealtissue) has flat parallel anterior and posterior surfaces, but the shapeof the organic cross-linked corneal implant 858 can be modified using aspecially-shaped polymeric back support structure. For example, as shownin FIGS. 69A-69C, the polymeric back support structure may have a flatstructure 864 (see FIG. 69A) for maintaining the flat shape of theorganic cross-linked corneal implant 858, a convex structure 866 (seeFIG. 69B) for giving the organic cross-linked corneal implant 858 aconvex shape, or a concave structure 868 (see FIG. 69C) for giving theorganic cross-linked corneal implant 858 a concave shape. Also, thesethin-walled transparent polymeric structures can have an index ofrefraction similar to the cornea (e.g., 1.3).

In one embodiment, the polymeric supporting elements are made fromhydrogel with a very high water content between 50-90% of water. Thishybrid implant can be used after a LASIK procedure or small incisionintrastromal inlay implantation, the structure can remain in place orremoved from the eye by forceps.

In one embodiment, all implants used in conjunction with a LASIKprocedure have no refractive surfaces initially, except for an index ofrefraction which is exactly as the corneal tissue, but after positioningover the exposed corneal stroma and modification of its surface with anexcimer laser and wavefront technology to correct all refractive errorsonly the anterior surface of the inlay is changed during the refractivesurgery in vivo, before the corneal flap is repositioned over the inlay.This in vivo correction of refractive error has the advantage that theposition of inlay over the corneal stroma does not change after thesurgery, since the inlay remains in place all the time during theexcimer laser ablation and the flap prevents its motion postoperatively,that is why one can achieve very high satisfaction after LASIK surgeryto >98% of the patients. Therefore, there will not be misplacement ofthe direction of the corrected astigmatism postoperatively which canhappen if the inlay surface(s) were corrected ex vivo, and then moved onthe cornea or inside in the corneal stroma.

In one embodiment, the crosslinker can be any crosslinker, butpreferably it is riboflavin which is excited by UV radiation of 350-390nm or a 370 nm to 390 nm laser wavelength which crosslinks all theproteins present in the implant including the potential pathogensincluding viral, bacterial, fungal and parasitic ones.

In one embodiment, the crosslinker is in a nanoparticle form conjugatedwith cell penetrating peptides (CPP) that penetrate all cellularmembranes and cross-link the intercellular proteins eliminating thecells of the human corneal inlay, thus preventing an immune responsefrom the tissue surrounding the inlay that can incite an antigenicresponse toward a corneal transplant.

In one embodiment, the combination of a riboflavin nanoparticle solutionwith CPP and an antibiotic, antiviral, antifungal, or anti-parasiticmedication is used as topical drops applied to the cornea, thenirradiated with UV light to cross-link the infected part of the cornea,so as to kill the pathogens and treat the infectious keratitis.

In one embodiment, all implants are prepared as described, but afterimplantation of the inlay, the riboflavin is injected in the cornealstromal cavity with a fine needle so that the riboflavin penetrates thewall of the stromal cavity around the implant for a distance of at least20 microns deep on each side that is cross-linked after termination ofthe surgery with external UV radiation for short period of time or 1-5minutes without crosslinking any other part of anterior or posteriorstroma not exposed to the riboflavin. The crosslinking of the wall ofthe cavity kills the stromal cells in that area and creates an immuneprivileged cavity that prevents an immune response to the implant, thein short situ cross-linking kills potential pathogen that may havecontaminated the inlay during the transport.

In one embodiment, medication is present in the riboflavin solution, andthe riboflavin nanoparticle solution, and the solution contains free orpolymeric slow release nanoparticles of an anti-inflammatory agents suchas NSAIDs, Rock inhibitors such as Fasudil, netarsudil, SAR407899,botulinum toxin at 1-10 international units, etc., anti-integrins,resiteganib, Natalizumab at microgram/ml, MLN-00002, Firategrast,IVL745, antagonists of αvβ3 and/or αvβ5 integrins, LM609, Vitaxin,Abegrin, CNTO95, Cilengitide etc wnt inhibitors such as Ant1.4Br/Ant1.4Cl, ivermectin, niclosamide, apicularen and bafilomycin, XAV939,XAV939, G007-LK and G244-LM, NSC668036, SB-216763, gemtuzumab, GSKinhibitors, such as SB-216763, etc., anti-VGEF avastin, lucentis,bevacizumab, afilbercept, antibiotic moxiflacin etc., or antivirals,valcyclovir etc. antifungal amphotericin B, voricanozole oranti-parasitic agents, such as niclosamide, ivermectin, etc. atnon-toxic concentrations alone or in combination. These compoundsparticularly Rock inhibitors and GSK inhibitors encourage sprouting ofthe cut corneal nerves and regeneration of nerves, and also can be usedin combination with nerve growth factors and Brimonidine as topical orslow release polymeric implants, incorporated in the inlay or placed aspolymeric implant, in the anterior chamber of the eye, in the vitreouscavity of the eye, under the conjunctiva, etc. (e.g., such as in thesubconjunctival drug delivery implant 722 shown in FIG. 46).

In one embodiment, the donor corneal tissue is cut with a circulartrephine of at least 7-8 mm in diameter, separating the circular cornealarea from the donor eye and removing the corneal epithelium and cornealendothelial cells from the donor corneal tissue, and cutting in twocircular leftover corneal donor portions of two equal dimensions with akeratome or a femtosecond laser, one portion from the anterior surfaceto a distance of 50-200 microns uniform thickness with Bowman'smembrane/stroma 3-D inlay, and another portion from the posteriorcorneal surface with a 50-200 microns uniform thickness having theDescemet membrane to build a 3-D circle of the corneal stroma andDescemet inlay, and then cross-linking portions with a solution havingriboflavin nanoparticles and an antibiotic, and irradiating the portionswith UV radiation to kill the stromal keratocytes along with potentialpathogens while crosslinking the collagen fibrils in the inlays, thentransplanting the cross-linked inlay over the exposed corneal stromaafter creation of a LASIK hinged flap to ablate the front surface of theinlay with an excimer laser using wavefront technology, and finallyreplacing the LASIK flap over the inlay covering it with the cornealstroma from all sides.

In another embodiment, the donor corneal tissue is cut with a circulartrephine of at least 7-8 mm in diameter, separating the circular cornealarea from the donor eye, and removing the corneal epithelium and cornealendothelial cells from the donor corneal tissue, and cutting in twocircular leftover corneal donor portions into two equal dimensions witha keratome or a femtosecond laser, one portion from the anterior surfaceto a distance of 50-200 microns uniform thickness with Bowman'smembrane/stroma 3-D inlay, and another portion from the posteriorcorneal surface with a 50-200 microns uniform thickness having theDescemet membrane to build a 3-D circle of the corneal stroma andDescemet inlay, and then cross-linking the portions with a solutionhaving riboflavin nanoparticles and an antibiotic, irradiating theportions with UV radiation to kill the stromal keratocytes along withpotential pathogens while crosslinking the collagen fibrils in theinlays, modifying the front surface of the inlays with an excimer laserto a desired shape, and then cutting the inlay with its Descemetmembrane in two or three sections to be implanted inside the hostcorneal stromal. In this embodiment, the Bowman's membrane and/orDescemet's membrane is kept so as to contribute to biomechanicalstability of the inlay. A pocket is created with a femtosecond laser,and then the inlay pieces are inserted through a small incision tocorrect the refractive power of the host eye. The inlay can be cut into2-3 pieces with a knife prior or after crosslinking the inlay.

In another embodiment, only a single inlay is cut from the donor cornea,rather than the multiple inlay portions described above.

In one embodiment, the corneal endothelium and epithelium of a humandonor cornea is removed mechanically or in combination with lowconcentration of alcohol in water, etc. prior to implantation of thehuman donor cornea.

In one embodiment, a uniform rectangular or circular piece of humandonor stromal tissue, a tissue culture grown cornea, a geneticallymodified cornea, or an acellular human or animal cornea is cut witheither a microkeratome or a femtosecond laser or a trephine to create a3-D corneal stromal circle with a thickness of 10 to 50 microns, 50 to150 microns, or 50 to 200 microns under sterile conditions (see FIG.70). The acellular human or animal cornea may be created using 0.05-0.5%or more sodium dodecyl sulfate (SDS), Triton X-100, benzalkoniumchloride (BAK), Igepal, or genipin for 15 minutes to 6 hours or moredepending on the concentration used to generate acellular cornealinlays, and the corneal tissue is cleaned in 100% glycerol, andsterilized by killing bacteria, viruses and/or parasites, or the cornealinlay may be re-sterilized by ethylene oxide or during the surgery bycrosslinking the donor tissue with the host cornea with UV light and0.1% of riboflavin nanoparticles.

In one embodiment, the human corneal inlay is made acellular by glycerinand cryopreservation and then thawing it for use in a physiologicalsolution of 0.1% riboflavin nanoparticles, and finally cross-linking itwith UV radiation after implantation.

In one embodiment, 0.1% of riboflavin nanoparticles in a physiologicalsolution of pH 6.5-7.5 and osmolality of about 300 milliosmole (mOsm)with or without antibiotics or antivirals, etc. as additives, appliedtopically on the cornea, the conjunctiva, and/or the lid margin andexposed to UV light or just the sunshine or a white light can sterilizethe infected cornea, conjunctiva, or the lid, while repeated daily onetime administration can cross-link the anterior part of cornealcollagen, etc. preventing corneal ectasia, progression of keratoconus,or high myopia.

In one embodiment, by using a trephine, the donor cornea is cut tocreate inlay circles of 3 to 9 millimeters (mm) or more in diameter forintrastromal implantation that can be preserved in a tissue culture or aphysiological solution having an antibiotic, antifungal, and apreservative or detergent, such as sodium dodecyl sulfate (SDS), toeliminate the contaminated pathogens. In addition, the donor cornea maybe stored in a low concentration of chondroitin sulfate, hyaluronicacid, or albumin to exert an oncotic pressure on the cornea so as toprevent it from swelling during the transport thereof.

In one embodiment, the corneal stromal inlay is de-cellularized with asolution of 0.1-0.5% sodium dodecyl sulfate (SDS) for simultaneouspreservation of the corneal inlay and killing the bacteria, viral, orparasites prior to implantation inside a corneal LASIK flap, thenplacing the corneal inlay over the exposed corneal stroma after cleaningit in 100% glycerol, correcting the refractive error of the host eyewith an excimer laser, then applying a solution of riboflavin/medicationover the inlay, replacing the flap, and crosslinking the inlay and thestromal wall with UV radiation.

In one embodiment, the corneal stromal inlay is de-cellularized with asolution of 0.1-0.5% sodium dodecyl sulfate (SDS) for simultaneouspreservation of the corneal inlay and killing the bacteria, viral orparasites prior to implantation inside a corneal pocket created with afemtosecond laser, correcting the refractive error of the inlay prior toimplantation in the host eye with the excimer laser or a femtosecondlaser, then the corneal inlay is placed inside a corneal pocket, and asolution of 0.1% riboflavin/medication in a hyaluronic acid solution isapplied to the inlay and the surrounding tissue, excess hyaluronic acidis removed from the cavity, and the inlay and the surrounding wall ofthe cornea are cross-linked with UV radiation.

In one embodiment, the stromal inlays are preserved in a hypothermicstorage at 2 to 8° C., or stored by freezing, by freeze drying, inglycerol, and/or gamma radiated and placed in hypothermic solution oftissue culture medium or organ culture medium.

In one embodiment, hypothermia reduces the demand for metabolic energyand since the stromal inlay does not have a corneal epithelium andendothelium or keratocytes its demand is very low, therefore it can bepreserved longer (e.g., 45 days or more) than a normal cornea can bepreserved, which is about one month.

In one embodiment, the preserving solution for the stromal inlay canhave chondroitin sulfate to prevent swelling of the tissue or it canhave 4-8% dextran 40, or include ascorbic acid, albumin, hyaluronicacid, or low molecular weight heparin, etc.

In one embodiment, the solution can become a gel with the corneal inlayin it by the addition of a crosslinker to it so as to crosslink thealbumen or adding trehalose to the solution and to maintain a gel-likestructure with proper pH and osmolality of the surrounding inlay.

In one embodiment, the preserving solution can be McCarey-Kaufman medium(M-K medium) containing 5% dextran 40.

In one embodiment, the storage temperature can be 2-4 degrees C. ormore, and the storage can be 30-45 days or more.

In one embodiment, the solution can have non-ionic surfactants(Poloxamer 188) for hypothermic storage.

In another embodiment, the intrastromal inlay can be placed in in 10-80ml Eagle's medium containing HEPES buffer, 26 mmol/l, NaHCO₃, 2% FBS, 2mmol/l L-glutamine, penicillin, streptomycin and amphotericin B, andstored at 4-34° C. for 30-45 days in a sterile container.

In one embodiment, the intrastromal inlays can be stored in a solutionof amphotericin B Optisol-GS that is effective against Candida species.

In one embodiment, the intrastromal inlay is transplanted over thecenter of an exposed corneal stroma which is created in a knownlaser-assisted in situ keratomileusis (LASIK) procedure using afemtosecond laser or a microkeratome where the flap is created andhinged away to expose the corneal stroma (see FIGS. 71-73).

In one illustrative embodiment, with reference to FIGS. 70-75, a methodof corneal implantation with cross-linking will be described. Initially,referring specifically to FIG. 72, a LASIK flap 914 is formed in acornea 912 of an eye 910 so as to expose a stromal tissue 916 of thecornea 912 underlying the flap 914. Then, the flap 914 is pivoted so asto expose the stromal tissue 916 of the cornea 912 underlying the flap914 (see FIG. 72). After pivoting the flap 914, an implant or inlay 918is inserted under the flap 914 so as to overlie the stromal tissue 916of the cornea 912 (see FIG. 73). Then, as shown in FIG. 73, laser energy920 (e.g., by means of an excimer laser) and/or microwaves are appliedto the implant 918 in the eye 910 so as to modify the refractive powerof the implant 918 while being monitored using a Shack-Hartmannwavefront system so as to achieve a desired refractive power for theimplant 918. Turning to FIG. 74, after the laser application, across-linking solution 922 that includes a photosensitizer is applied tothe implant 918 (e.g., by drops or injection). Next, the implant 918 iscovered with the flap 914 such that the implant 918 is surroundedentirely by the stromal tissue of the cornea 912. Finally, as shown inFIG. 75, the implant 918 is irradiated (e.g., with ultraviolet (UV)radiation 924) so as to activate cross-linkers in the implant 918, andthereby cross-link the implant 918 to prevent an immune response to theimplant 918 and/or rejection of the implant 918 by the patient. The wallof the stroma of the eye 910 surrounding the implant 918 may also becross-linked together with the implant 918 for a predetermined timeduration (e.g., ten minutes).

In the illustrative embodiment of FIGS. 70-75, the implant or inlay 918may be formed from donor corneal tissue, a tissue culture grown cornea,or a 3-D printed cornea. When the implant or inlay 918 is formed fromdonor corneal tissue, the donor corneal tissue may initially be cut intoa circular implant 926 using a circular trephine (see FIG. 70), wherethe circular implant has a diameter between 3 millimeters and 10millimeters. Then, the circular implant 926 may be cut into two circulardisks using a keratome or a femtosecond laser, each of the two circulardisks having a thickness between 50 microns and 200 microns.

In one embodiment, the stromal inlay size and thickness is chosendepending on the degree of the corrective dioptric power that is needed.In one embodiment, an inlay with a thickness of 50 microns can correct 5dioptric powers, and with 150 micron thickness can correct 15 dioptricpower, etc.

In one embodiment, the stromal inlay has a diameter of about 3 mm and isplaced just inferior to the center of the visual axis and corrected tohave a dioptric power of ±1-3.00 D or more for patients with presbyopiafor reading that is creating a bifocal cornea, where the patient seesfar through the center of the cornea and near, through the small inlay.

In one embodiment, the refractive error of the eye is corrected afterthe LASIK flap is created and the inlay is positioned over the exposedcenter of the corneal stroma by the use of an excimer laser andwavefront technology and a Shack-Hartmann sensor to correct myopic,hyperopic, or astigmatic or other higher order of refractive errors ofthe eye (see FIG. 73), then a few drops of the solution of 0.1%riboflavin nanoparticles, conjugated with cell penetrating peptides(CPP) or activatable cell penetrating peptides (ACPP) having antibiotic,antifungal, or anti-parasitic medication, etc. or anti-inflammatoryagents, such as Rock inhibitors, anti-integrins, GSK inhibitors, etc. ata non-toxic dose of 1 microgram to 100 microgram in 0.1 milliliters (ml)or a concentration for use in intravitreal irrigating solutions, or asslow release biodegradable polymeric polylactic acid or polyglycolicacid or their combinations of porous silicon, micelles orpolycaprolactone nanoparticles are administered over the inlay andexposed corneal stroma (see FIG. 74) to treat the inflammatory processesand prevent tissue scarring.

In one embodiment, the riboflavin may penetrate the inlay within 1-3minutes.

In one embodiment, the hinged corneal flap is repositioned so that theinlay is covered on all sides by the host corneal stroma, which now isalso exposed the riboflavin solution.

In one embodiment, the nanoparticles of riboflavin solution can beinjected under the corneal flap over the inlay though a small 25-30gauge needle or smaller needle to expose the inlay and the cornealcavity to the riboflavin solution, then the cornea, including the inlayis irradiated with UV radiation of 3-10 milliW/cm² from outside thecornea for a short period of 3-10 minutes to cross-link only the inlayand its surrounding corneal stroma, thereby killing all remaining cellsin the inlay and any potential pathogens that might have contaminatedthe inlay or the stroma by the combination effects of the UV radiation,riboflavin nanoparticles, CPP, and medication and sterilize the inlayand the stromal cavity. In one embodiment, the corneal cavity and theflap can be irradiated with UV laser brought under the corneal flapthrough a fiber optic, thus crosslinking the inlay and the surroundingstromal tissue while the tissue close to the corneal endothelium orclose to the anterior surface of the corneal are not cross-linked.

In one embodiment, the nanoparticles of riboflavin solution can beinjected under the corneal flap over the inlay though a small 25-30gauge needle or smaller needle to expose the inlay and the cornealcavity to the riboflavin solution, then the cornea, including the inlayis irradiated with UV radiation of 3-10 milliW/cm² for a short period of5-10 minutes using a fiber optic with an small lens on its tip to focusthe laser beam only for a distance of 20-50 microns, and then itdiffuses out so that the endothelial cells, or the crystalline lens, orthe retina is not exposed to a heavy dose of UV radiation. In oneembodiment, the tip of the fiber optic moves all over the front surfaceof the intrastromal inlay to crosslink only the inlay and itssurrounding corneal stroma, killing all remaining cells in the inlay andany potential pathogens that might have contaminated the surgical areaby the combination effects of the UV radiation, riboflavinnanoparticles, CPP, and medication and sterilize the inlay and thestromal cavity while eliminating the toxic effect of the UV radiation tothe anterior or the posterior corneal tissue.

In one illustrative embodiment, with reference to FIGS. 76-81, anothermethod of corneal implantation with cross-linking will be described.Initially, referring specifically to FIGS. 76 and 77, an intrastromalcavity 932 is formed in the cornea 930 of an eye 928. Then, theintrastromal cavity 932 of the cornea 930 is filled with a shaped donorcornea implant 934 (see FIG. 78). After inserting the shaped donorcornea implant 934 in the intrastromal cavity 932, a cross-linkingsolution 936 that includes a photosensitizer (e.g., riboflavin) isinjected into the implant 934 (e.g., by using a needle for injection—seeFIG. 79). Finally, as shown in FIG. 80, the implant 934 is irradiated(e.g., with ultraviolet (UV) radiation 938) so as to activatecross-linkers in the implant 934, and thereby cross-link the implant 934to prevent an immune response to the implant 934 and/or rejection of theimplant 934 by the patient. The wall of the stroma of the eye 928surrounding the implant 934 may also be cross-linked together with theimplant 934 for a predetermined time duration (e.g., ten minutes). InFIG. 80, the ultraviolet (UV) radiation 938 is shown being externallyapplied to the cornea 930 of the eye 928 with the implant 934. Although,rather than being externally applied, the ultraviolet (UV) radiation mayalso be internally applied to the implant 934 by means of a fiber optic940 (see FIG. 81). For example, the UV radiation may be applied insidethe tissue with the fiber optic 940, which may include a small incisionlens at its tip to focus the lens in the tissue for a very shortdistance of about 100 microns and then it diverges, thus preventingdamage in the back of the corneal, endothelial cells, the crystallinelens, the retina, the corneal epithelial cells, or the corneal nervesplexus under the epithelium.

In the illustrative embodiments described above, the intrastromalimplant or inlay 918, 934 is not cross-linked, and its refractive poweris not corrected prior its transplantation into the recipient cornea912, 930. The implantation procedure may be either a MESICK(Meso=inside, I=inlay, C=Crosslinking, K=Keratomileusis) procedure, orsmall incision implant procedure. In one or more other embodiments, therefractive power of the implant or inlay is corrected, but cross-linkingis done after implantation inside a stromal pocket.

In one embodiment, microwave radiation may be used to modify therefractive error of the cornea or the inlay rather than laser radiationdelivered by an excimer laser. Also, nanojoule pulses of a femtosecondlaser may be applied to small lenticules after implantation to changethe index of the refraction of the small incision lenses in the stromalcavity. In addition, a combination of cross-linking and microwaveapplication may be used to crosslink the cornea and correct therefractive error while maintaining the corneal transparency.

In one embodiment, subsequently an antibiotic ointment or solution isapplied to the operated eye in the post-operative period, and in someembodiments, if needed, the crosslinking process can be repeated insidethe stroma and in the inlay to kill potential invading cells as neededand to keep the inlay's transparency.

In one embodiment, after the surgery, the refractive power of the corneaor the inlay can be modified using focally administrated focusedmicrowaves.

In one embodiment, the corneal crosslinking can be combined withmicrowave application in the same session to achieve synergisticresults.

In one embodiment, one can modify the refractive index of the cornealinlay by the use of few nanojoules of femtosecond laser applicationwhich does not cut or damage the inlay.

In one embodiment, the crosslinking solution and its components are usedto treat an infected wound of the cornea, skin, or mucosa to beirradiated with UV light and kill the bacterial, viral, fungal and/orparasitic infection or the antibiotic resistant bacteria MRSA, orviruses or prions in the infected tissue and the corneal implant, etc.

In one embodiment, the implant is formed from donor corneal tissue, andwherein, prior to the step of inserting the implant under the flap, themethod further comprises the steps of cutting a circular implant from adonor cornea using a circular trephine, the circular implant having adiameter between approximately 3 millimeters and approximately 9millimeters or more; and cutting the circular implant disks using atrephine or a femtosecond laser, the two circular disks having athickness between approximately 50 microns and approximately 100 micronsand shaping the inlay surface's refractive power with an excimer laser.

In one embodiment, a corneal pocket is created in the host cornealstroma with a diameter of 3-9 mm slightly larger than the diameter ofthe inlay for ease of implantation, using a femtosecond laser, andmarking its center and making a small side incision in the cornea toaccess the inside of the intrastromal pocket and to separate the stromalpocket using a micro-spatula or a bent needle and hyaluronic acid, andthen a pre-shaped corneal inlay and hyaluronic acid is implanted aloneor with N-Vinylpyrrolidone and riboflavin for ease of implantation inthe corneal stromal pocket, riboflavin or photosensitizer nanoparticlesare injected in the corneal pocket to penetrate the inlay and slightlythe wall of the pocket, and then the inlay and the wall of the pocketand hyaluronic acid are cross-linked with UV radiation, thus convertingthe hyaluronic acid into a gel.

In one embodiment, one uses a fluid containing inorganic or organicmolecules such as hyaluronic acid, collagen, elastin, chondroitinsulfate or hydrogel, polyethylene glycol, or biotin, polycaprolactone,chitosan, etc. in various combinations in a physiologic solution havingin addition a crosslinker such as riboflavin, N-Vinylpyrrolidone, orother medications.

In another embodiment, the implant is formed from donor corneal tissueor a 3-D printed cornea, and prior to the step of inserting the implantin the corneal stroma, one performs the steps of cutting a circularimplant from a donor corneal stroma, etc. using a circular trephine, thecircular implant having a diameter between approximately 3 millimetersand approximately 9 millimeters and creating a corneal pocket in thehost corneal stroma with a diameter of 4-10 mm using a femtosecondlaser, or any other laser and making one or two small side incisions inthe cornea with the same femtosecond laser or a knife, to access theinside of the intrastromal pocket and to separate the walls of thepocket using a micro-spatula and hyaluronic acid and cutting thecircular implant into two concentric circular parts, one circular diskinside the outer doughnut part using a smaller trephine than thediameter of the inlay or a femtosecond laser, the two concentriccircular parts having a thickness between approximately 50 microns toapproximately 100 microns and their surfaces are shaped to form adesired refractive power with an excimer laser to correct a myopic,hyperopic or astigmatic error of the eye after their separateimplantation using forceps or an injector, the annular part first, thenthe disc part through a small incision that is made from outside toaccess the inside of the stromal pocket where the two pieces are joinedagain after their implantation. The pre-shaped corneal inlay isimplanted with the annular part first, then the disk inside it secondalong with hyaluronic or low molecular weight heparin for ease ofimplantation in the corneal stromal pocket, riboflavin or riboflavinnanoparticles are injected as a 0.1-1% solution or anotherphotosensitizer is injected along with other antibacterial or antifungalmedications in the corneal pocket to penetrate the inlay and slightlythe wall of the pocket, and finally the inlay and the wall of the pocketare crosslinked with UV radiation and to kill the potential pathogeniccontamination associated with the implant.

In one embodiment, the inner disk implant can be organic and the outerannular part of the implant can be made from a synthetic transparentpolymer or acrylic that is resilient or flexible and in one piece ormultiple separate pieces, which may be implanted through a smallincision made in the wall of the cornea after creating a corneal pocketwith a larger diameter for the desired implant for ease of implantation.

In one embodiment, one uses a fluid containing inorganic or organicmolecules, such as hyaluronic acid, low molecular weight heparin,collagen, elastin, chondroitin sulfate, hydrogel, polyethylene glycol,chitosan, biotin, polycaprolactone, etc. or in various combinations in aphysiologic solution having, in addition to a crosslinker such asriboflavin or N-Vinylpyrrolidone, other medications, such as Rockinhibitors or anti-integrins, where the transparent solution has arefractive index of equal to 1.34 or more for correction of a hyperopicerror, and is to be injected into a corneal stromal pocket of therecipient cornea prepared using a femtosecond laser or picosecond laser,etc. and to increase the anterior curvature of the stromal cavity bypushing the anterior wall of the cavity forward after injecting moresolution, or reducing its curvature by removing an amount of fluid fromthe stromal cavity, to treat a hyperopic refractive error to the desireddegree using a wavefront technology during the processes of theinjection and immediately irradiating the cornea with UV radiation toconvert the fluid-filled cavity into a cross-linked gel creating anintrastromal transparent lens with an anterior convex surface and anindex of refraction of 1.34 or more and cross-linked adjacent cornealstromal tissue to prevent an immune response to the gel and killpotential existing pathogens in the gel and the cornea and prevent thelens from extruding through the small stromal opening made forinjection.

In one embodiment, the crosslinking of the cornea and the inlay can berepeated using an internal injection of riboflavin nanoparticles in aphysiological solution in the stromal cavity or applyingriboflavin/medication drops from outside to penetrate the anteriorcornea and the inlay, which are being cross-linked again with externallyapplied UV radiation to kill potential invading inflammatory cells, ororganisms if any sign of rejection or cellular invasion or infection isobserved to stop the process, or the implant is emulsified with afemtosecond laser and washed out or removed or replaced.

In one embodiment, a corneal pocket is created inside the corneal stromausing a femtosecond laser and a circular 3-D circular area is cut with auniform thickness of approximately 60 microns inside the pocket prior toseparation of the anterior stromal layer, then a small incision is cutto the side of the cornea to access inside the corneal pocket and removethe circular uniform disc from the stroma near the back of the cavity tocreate more space inside the corneal pocket and a a new corneal inlay isplaced inside the pocket, the inlay having a corrected refractive errorof the eye for hyperopia, myopia, and astigmatisms, and riboflavinnanoparticles along with hyaluronic acid in solution is injected insidethe corneal cavity, and the inlay and the surrounding stromal tissue iscross-linked with UV radiation or another wavelength of light if adifferent crosslinker is used, to prevent corneal rejection orinfection.

In another embodiment, a cavity is formed and a uniform disk oroval-shaped portion of the stroma of about 60-100 micron tissuethickness is removed, and the cavity is injected with cross-linkablemolecules in a transparent biocompatible solution having a crosslinkerto correct the hyperopic, hyperopic, or astigmatic refractive error ofthe eye, and the cross-linkable fluid is cross-linked by externallyapplied UV laser radiation to cross-link the fluidic cavity and convertit into a cross-linked gel and cross-link the corneal pocket wall, whilekilling potential pathogens infecting the surgical area and preventingthe escape of the gel inlay from the small incision that was initiallymade to access the corneal cavity and preventing glare seen afterremoval of the stromal tissue.

In one embodiment of corneal implantation with cross-linking, the methodcomprises the steps of: (i) forming a flap in a cornea of an eye so asto expose a stromal tissue of the cornea underlying the flap; (ii)pivoting the flap so as to expose the stromal tissue of the corneaunderlying the flap; (iii) inserting an implant under the flap so as tooverlie the stromal tissue of the cornea; (iv) applying laser energy tothe implant in the eye so as to modify the refractive power of theimplant and the eye while being monitored using a Shack-Hartmannwavefront system so as to achieve a desired refractive power for theimplant.

In one embodiment, a cross-linking solution that includes aphotosensitizer is applied to the implant and to the stromal tissue ofthe cornea surrounding the implant, the implant is covered with theflap, the implant being surrounded entirely by the stromal tissue of thecornea; and the implant and the stromal tissue of the cornea surroundingthe implant is irradiated so as to activate cross-linkers in the implantand in the stromal tissue of the cornea surrounding the implant, andthereby cross-link the implant and the stromal tissue of the corneasurrounding the implant to prevent an immune response to the implantand/or rejection of the implant by the patient.

In one embodiment, the implant is formed from donor corneal tissue, atissue culture grown cornea, a 3-D printed cornea, or preferably fromhuman donor corneal tissue that is stored in a sterile container havingan antibiotic and a preservative solution without cross-linking theimplant prior to implantation, and a corneal LASIK flap is formed in therecipient cornea using one a femtosecond laser or a mechanical keratome.

In one embodiment, the photosensitizer of the cross-linking solutioncomprises nanoparticles of riboflavin, or another photosensitizer, andthe step of irradiating the implant comprises irradiating the implantwith ultraviolet light or a chemical crosslinker.

In one embodiment, the cross-linking solution contains one or moremedications for treating a medical condition of the patient, the one ormore medications selected from a group consisting of a Rock inhibitor,Wnt inhibitor, an integrin inhibitor, a GSK inhibitor, an antibiotic, ananti-fungal medication, and combinations thereof.

In one embodiment, the correction of the refractive power of the eye isdone after placement of the inlay over the exposed corneal stroma usingan excimer laser so as to modify the refractive power of the implant andthe eye under the control of wavefront technology controlled with aShack-Hartmann sensor.

In one embodiment, the cross-linking solution includes a photosensitizerin the form of riboflavin nanoparticles and a medication selected fromthe group consisting of an antibiotic, an antiviral, an antifungal, ananti-parasitic, and combinations thereof; and the cornea is irradiatedwith ultraviolet light to cross-link an infected part of the cornea soas to kill pathogens and treat infectious keratitis.

In one embodiment, the implant is formed from donor corneal tissue bycutting one or more circular implant disks from a donor cornea using atrephine or a femtosecond laser so as to form the implant, the one ormore circular implant disks having a diameter between approximately 3millimeters and approximately 9 millimeters, and the one or morecircular implant disks having a thickness between approximately 50microns and approximately 100 microns; and a surface of the one or morecircular implant disks is shaped using an excimer laser prior toimplantation under a corneal flap or inside a corneal stromal cavity.

In one embodiment, the implant and the wall of the corneal cavity iscross-linked after implantation using a fiber optic or the implant andthe wall of the corneal cavity contains a crosslinker that can beirradiated using an external energy source (e.g., UV radiation) throughthe surface of the cornea, and the anterior part of the cornea does nothave a crosslinker and is not cross-linked during this procedure.

In one embodiment, the cross-linking solution contains nanoparticles ofriboflavin, cell penetrating peptides, and an antibiotic, antifungal,anti-viral, and/or antiparasitic medication to treat a bacterialinfection of the cornea, skin, or mucosa or the eyelids with ultravioletradiation to kill the invading pathogens.

In one embodiment, a corneal cavity of 3-9 mm wide or larger is createdin the host corneal stroma using a femtosecond laser where a small sideincision is made in a recipient cornea of an eye of a patient forinsertion of a circular implant into the stromal pocket by injecting thecross linking solution, and then crosslinking the inlay and the wall ofthe cavity so as to prevent an immune response to the implant and/orprevent rejection of the implant by the patient's tissue.

In one embodiment, the step of forming the small side incision in therecipient cornea of the eye further comprises making the small sideincision in the recipient cornea with a femtosecond laser or a knife.

In one embodiment, a cavity is formed in the host stroma and the wall ofthe cavity is separated using a spatula or a bent needle while injectionof a solution of hyaluronic acid, and an implant is inserted along withinjection of riboflavin nanoparticles to crosslink the inlay and thewall of the cavity and the remaining hyaluronic acid irradiating thecircular implant with ultraviolet light so as to cross-link the circularimplant and the portion of the bounding wall of the pocket and thehyaluronic acid, and kill any potential pathogenic contaminantsassociated with the circular implant and create a clear tissuetransition preventing postoperative glare.

In one embodiment, the circular implant is cut with a circular trephinehaving an outer diameter between approximately 3 millimeters andapproximately 9 millimeters; then the circular implant is cut into twoconcentric circular parts, the first one of the two concentric circularparts comprising an annular disk portion and the second one of the twoconcentric circular parts comprising a central disk portion inside theannular outer portion, the central disk portion being cut using afemtosecond laser or a trephine having a diameter smaller than the outerdiameter of the circular implant, each of the two concentric circularparts having a thickness between approximately 30 microns andapproximately 100 microns.

In one embodiment, the surface of the implant is shaped with an excimerlaser to prior to implantation in the corneal stromal pocket to form adesired refractive power so as to correct a myopic, hyperopic, orastigmatic error of the eye.

In another embodiment, the implant inserted into the pocket of therecipient cornea via a small incision by initially inserting the annulardisk portion into a pocket via the small incision using an injectorsyringe or a forceps, and then subsequently inserting the central diskportion into the pocket via the small incision using the injectorsyringe or the forceps, and joining the two concentric circular partstogether inside the pocket. Then, a crosslinker with hyaluronic acid isinjected inside the pocket over the implant, and the implant and thestromal wall are cross-linked with UV radiation to prevent postoperativeglare.

In one embodiment, the central disk portion of the circular implant isorganic and the annular disk portion of the circular implant is madefrom a synthetic transparent polymer, acrylic, or hydrogel, etc. that isresilient or flexible.

In one embodiment, a corneal pocket is formed in the host's posteriorcorneal stroma using a femtosecond laser, first a circular oroval-shaped three-dimensional disk is cut in the corneal stroma, and asecond cut is made in front of the first cut, but the second cut isslightly larger with a substantially uniform thickness or distance fromthe first cut of approximately 60 microns and 100 microns using thefemtosecond laser so as to form the pocket, and the circular oroval-shaped three-dimensional posterior disk is removed from the hostcorneal stroma to create more space inside the pocket; and the anteriorlamella is separated and the posterior lamellar is removed using amicro-spatula or a bent needle, hyaluronic acid is applied for ease ofremoval of the tissue and insertion of the circular implant into thepocket in the recipient cornea of the eye and to smoothen up the wall ofthe cavity; riboflavin or other photosensitizer nanoparticles areinjected into the pocket in the recipient cornea of the eye so as topenetrate the circular implant and a portion of a bounding wall of thepocket; and the circular implant is irradiated with ultraviolet light oranother wavelength of light so as to cross-link the circular implant andthe portion of the bounding wall of the pocket, and to prevent cornealrejection of the circular implant and/or prevent corneal infection andpostoperative glare.

In one embodiment, a corneal pocket is formed in the host's posteriorcorneal stroma using a femtosecond laser, first a circular oroval-shaped three-dimensional disk is cut from the corneal stroma, thena second cut is made in front of the first cut, but the second cut isslightly larger with a substantially uniform thickness or distance fromthe first cut of approximately 60 microns and 100 microns using thefemtosecond laser so as to form the pocket, and the circular oroval-shaped three-dimensional posterior disk is removed from the hostcorneal stroma to create more space inside the pocket by creating twosmall side incisions opposite each other to gain entry in the pocketfrom two sides, thus simplifying removal of the stromal tissue orimplantation of an inlay afterwards having the two instruments insertedinside the corneal pocket, one having a sharp edge to separate thetissue from the underlying stroma, and the other to hold the edges ortwo prongs to pull the tissue for ease of manipulation and removal ofthe circular or oval-shaped three-dimensional disk with a uniformthickness from the host's posterior corneal stroma to create more spaceinside the pocket; and portions of the pocket are separated using amicro-spatula or a bent needle and hyaluronic acid; and hyaluronic acidis used for ease of insertion of the circular shaped implant into thepocket to correct the refractive error of the eye in the recipientcornea of the eye and smoothen up the wall of the cavity, an riboflavinor other photosensitizer nanoparticles are injected with or withouthyaluronic acid into the pocket in the recipient cornea of the eye so asto penetrate the circular implant and a portion of a bounding wall ofthe pocket; and the circular implant is irradiated with ultravioletlight or another wavelength of light so as to cross-link the circularimplant and the portion of the bounding wall of the pocket, and toprevent corneal rejection of the circular implant and/or prevent cornealinfection and postoperative glare.

In one embodiment, after creating and removing a circular or oval-shapedthree-dimensional disk from the pocket, cross-linkable molecules ormonomers are injected in a transparent biocompatible solution containinga crosslinker into the pocket so as to correct a hyperopic, hyperopic orastigmatic refractive error of the eye; and under control of wavefronttechnology and a Shack-Hartmann sensor, the transparent biocompatiblesolution is cross-linked by externally applying ultraviolet radiation soas to cross-link the solution inside the pocket and convert the solutionto a cross-linked gel, and to further cross-link a bounding wall of thepocket and kill any potential pathogens infecting the surgical area ofthe eye and preventing postoperative glare.

In one embodiment, one uses a femtosecond laser or picosecond laser toform the pocket in the recipient cornea of the eye, then injects across-linking solution into the pocket in the recipient cornea of theeye so as to penetrate the circular implant and a portion of a boundingwall of the pocket, the cross-linking solution comprising a transparentfluid that contains inorganic or organic molecules or monomers thatinclude hyaluronic acid, and its modified derivatives,N-isopropylacrylamide, alginate, thiol, tyramine, collagen, elastin,chondroitin sulfate, hydrogel, photo-crosslinked hydrogel, polyethyleneglycol, poly(vinyl alcohol) (PVA), biotin, chitosan, and combinationsthereof, in addition to a photosensitizer and one or more medications,the transparent fluid having a refractive index equal to 1.34 or morefor correction of a hyperopic error; increasing the corneal curvature bypushing an anterior wall of the pocket forward by injecting more of thetransparent fluid, or reducing an amount of the transparent fluid totreat a hyperopic refractive error to the desired degree using wavefronttechnology; and the recipient cornea is irradiated with ultravioletlight to convert the transparent fluid in the pocket into a cross-linkedgel, thus creating an intrastromal transparent lens with an anteriorconvex surface and an index of refraction of 1.34 or more, producingcross-linked adjacent corneal tissue so as to prevent an immune responseto the cross-linked gel and kill potential existing pathogens in thecross-linked gel and the recipient cornea, and to prevent theintrastromal transparent lens from extruding through the small sideincision in the recipient cornea and prevent postoperative glare.

In one embodiment, the crosslinking preparation containing medicationsis cross-linked and used as a device that can be placed over or underthe conjunctiva, or the lid, so as to be used as a drug delivery devicefor delivering a non-toxic dose of antibiotic, antiviral, antifungal,anti-parasitic, Rock inhibitors, integrin inhibitors, or Wnt inhibitorsafter LASIK surgery, corneal transplant, PRK, or after any corneal orconjunctival procedure to treat infection, reduce inflammatory processto reduce scarring, after ocular surgery, encourage nerve growth in thecornea after refractive surgery and treat infective diseases of thecornea, conjunctiva, or the lid.

In one embodiment, the photosensitizer or the cross-linking solution isriboflavin, and the one or more medications are selected from a groupconsisting of a Rock inhibitor, Wnt inhibitor, an integrin inhibitor, aGSK inhibitor, an antibiotic, an anti-fungal medication, andcombinations thereof.

In one embodiment, the recipient cornea and the intrastromal inlay arecross-linked one or more additional times with the crosslinker and asuitable medication is applied if any sign of rejection and/or cellularinvasion or infection is observed so as to stop the rejection and/or thecellular invasion.

In one embodiment, one can emulsify the intrastromal transparent gelusing a femtosecond laser, washing out or removing the emulsifiedintrastromal transparent gel, and replacing the emulsified intrastromaltransparent gel with a new intrastromal transparent gel-ablepreparation.

In one or more further embodiments described hereinafter, improvedprocedures for preserving donor corneas will be explained. Although thetransplantation of the cornea was introduced a century ago, it was muchlater in the 1940s-1950s that corneal preservation for the fullthickness transplantation was attempted. Using the so-called moistchamber, initially the entire eye was stored at 2-6° C. as hypothermic.Subsequently, the cornea and some surrounding tissues were kept in organculture at 31-37° C. Freezing the corneal tissue was discouraged, sinceit damages the endothelial cells that are useful for regulating thefluid transport in the full thickness corneal graft.

At present, the maximal time that the corneal tissue can be preserved isabout 14 days, but, in general, the preserved cornea loses theirviability after 3-4 days. These corneas become permeable to water in theculture media, become cloudy and useless as corneal transplant arediscarded.

In one embodiment, the corneas are obtained shortly after the donor'sdeath. Biopsies of the eye surfaces are taken for bacteriologicalevaluation and culture. Corneal storage after corneal or scleralcrosslinking does not require any matching, since all proteins, such ascollagen are cross-linked during the process of cross-linking using aphotosensitizer, such as riboflavin, and light, such as ultraviolet (UV)light and the cross-linked collagen is not immunogenic. Nevertheless,after the initial step of bacteriological biopsy, the eye is placed in abottle for transfer under aseptic conditions having a solutioncontaining antibiotics and/or antimycotics, since about 10% of the eyescan be contaminated, but the actual incidence of endophthalmitis is verylow.

In one embodiment, the retrieved corneas from the eye bank are preparedby dissecting the soft tissue conjunctiva, muscles, retina, choroid,iris, the ciliary body, and the optic nerve, which are cut away andremoved. The endothelial cells and the corneal epithelium are scrapedaway leaving only the sclera and the corneal stroma. The corneal stromaand scleral tissue are washed and cleansed in a physiological solutionor washed with a solution of povidone iodine and then with aphysiological solution, and prepared for in vitro crosslinking.

In one embodiment, the advantage of using a photosensitizer and itsactivation in the tissue with light (e.g. UV light) is that the cornealtissue is cross-linked, and it does not lose its transparency, which isvery important after corneal transplantation so that the light reachesthe retina without being distorted or absorbed, whereas thecross-linking of tissue with the use of chemical crosslinkers, such as aformaldehyde, interferes with not only the transparency of the cornea,but makes it very stiff, in addition to being very toxic to thesurrounding tissue.

In one embodiment, the cornea or the sclera are cut to a specific shapeand size as needed.

In one embodiment, the corneal tissue can be cut using a knife,keratome, a femtosecond laser, or excimer laser to the shape andthickness of a lens with defined diameter, or dioptric power creatingplus or minus astigmatic lens, or a toric lens.

In one embodiment, the corneal or scleral tissues are initiallycross-linked prior to the surgery, and then cut using a knife, keratome,a femtosecond laser, or an excimer laser to the shape and thickness of alens with defined diameter, or dioptric power and thickness creating aplus or minus astigmatic lens or a combination thereof.

In one embodiment, the corneas may be cut to be circular, with a centralhole of 1-7 millimeters (mm) or rectangular, oval, etc. or any desiredshape for implantation inside the host cornea in a pocket created with afemtosecond laser at any depth of the host corneal stroma.

In one embodiment, the lens implant may have one of: (i) a concavesurface to correct myopic refractive errors (i.e., a minus lens forcorrecting nearsightedness), (ii) a convex surface to correct hyperopicrefractive errors (i.e., a plus lens for correcting farsightedness), or(iii) a toric shape to correct astigmatic refractive errors.

In one embodiment, the corneas are cross-linked for storage in eye bankby placing them is a crosslinking solution having any photosensitizer,such as riboflavin, at any desired concentration and osmolarity,preferably of 300 mOsm/L at a pH of 4-8, preferably pH 7.4, and having aconcentration between about 0.1% to about 100%, for example, ofriboflavin therein, etc.

In one embodiment, the cross-linked cornea and sclera can be placed in asterile vial under vacuum, or in a standard storage solution, such astissue culture medium 199 (M-199) containing 5% dextran 40 forhypothermia, even cryopreservation, and can be kept frozen under verylow temperature after ice-free cryopreservation by vitrification of thecross-linked cornea in a high concentration of propane-1,2-diol orliquid nitrogen by stepwise cooling the tissue, etc.

In one embodiment, the cellular components of the corneal stroma may beinitially destroyed using enzymatic agents, such as Trypsin, Nucleases,Phospholiphases A₂ (PLA₂), Sera, all have shortcomings of damaging thebasement membrane, such as Bowman's or Descemet's membrane. Althoughdestruction of the cellular elements can be beneficial, alone it isuseless since the antigenic component of the stroma that is the collagenis not cross-linked.

In one embodiment, one can destroy the cellular component of the cornealor sclera initially using chemical means, such as Ethanol, Glycerol,acids and alkalis, Peracetic acid, Ammonium hydroxide ionic detergents,Sodium dodecyl sulfate, Sodium deoxycholate non-ionic detergents,zwitterionic detergents, (CHAPS), but all damage the Glycosaminoglycans(GAG) component of the stroma of the cornea or the sclera and do notcross-link the collagen, thus the tissue maintains its antigenicity.

In one embodiment, the corneal stroma or the sclera is exposed tohypo-and hypertonic solutions that are basically harmless, but it is noteffective in destroying the cellular elements of the cornea or scleraand does not cross-link the collagen, etc. while maintaining theantigenicity of the tissue.

In one embodiment, the cornea or sclera is exposed to Tris-HCL thateliminates cellular elements and also damages the GAG component of thetissue, and it does not cross-link the collagen.

In one embodiment, the physical means, such as freeze-thawing or snapfreezing can be used that damages the cellular elements of the tissue,but also damages the extracellular matrix (ECM) of the tissue withoutcrosslinking the collagen.

In another embodiment, the tissue can be exposed to high ultrasonicenergy and frequency that can damage the cells when the cornea isimmersed in the fluid and the ultrasonic transducer in also immersed inthe solution. This technique and/or exposing the cornea or sclera tohigh hydrostatic pressure can destroy the cells without damaging thecollagen in the stroma or in the sclera, but both techniques do notcross-link the cornea, and the tissues maintain their immunogenicity andcreate an immune response that encourage immune cell migration andenzymatic destruction of the implant, and the implant is ultimatelyrejected.

In one embodiment, one can expose the donor cornea or sclera toultrasonic destruction or high hydrostatic destruction of the cellularelements, then apply a photosensitizer, such as riboflavin, to thetissue immersed in a riboflavin solution that is then radiated with UVradiation or another light to crosslink the cornea or the scleraltissue. Subsequently, the tissue is cleansed in a saline solution towash out the reactive oxygen species and enzymes that are released bydestroying the cells by a simple agitation of the tissue or a lowfrequency ultrasound. In one embodiment, this ultrasonic technique canbe also applied to in vivo corneas or the sclera after theircross-linking with a photosensitizer/UV radiation.

In one embodiment, the cross-linked cornea can be used as an inlay toaugment the thickness of the cornea and render it more resilient forrefractive surgery procedures.

In one embodiment, the cross-linked cornea is placed over the hostcornea using a Shack-Hartmann system or other available instrument tocorrect the refractive error of the eye with an excimer laser, prior toimplantation of the implant inside a corneal pocket.

In one embodiment, the cross-linked cornea or sclera can be used toreplace injured lip, nose, or cheek augmentation in cosmetic surgery,chin augmentation, or reconstruction, while the cornea or sclera that iscross-linked maintains its flexibility and softness.

In one embodiment, for eye bank storage, the cornea and/or sclera can becross-linked (see e.g., FIG. 4B) using a photosensitizer/cross-linkerand UV radiation to kill the cellular elements and crosslink the corneaand scleral collagen making them less antigenic while being able tostore them for a long time (e.g., greater than 1 year storage) andmaintaining the corneal stromal clarity.

In one embodiment, the corneal stroma and/or the sclera can be preservedby the photosensitizer crosslinking technique and kept almostindefinitely if the cross-linked cornea is kept at temperature close tozero, and is sealed in a bottle under vacuum for many years. Thecross-linked corneal stroma can be washed with a sterile physiologicalsaline solution to regain its shape and transparency and shaped with afemtosecond laser to any shape lenslet for correction of myopia,hyperopia, and astigmatism while strengthening the cornea by increasingits shape rather than decreasing it as is done with the presentphotorefractive, or LASIK or SMILE laser procedures. The preservedcorneal inlay also can be once again cross-linked or exposed to UVradiation or gamma radiation to completely sterilize it. This procedureprevents post implantation bacteria, fungal parasites, or viralinfections seen often after implantation of the partial corneal tissueor inlay where the pathogens can hide in the crevices of the tissue(e.g., inside the cornea) and grow a few days or weeks later producing adifficult to manage endophthalmitis or corneal ulcer.

In one embodiment, the corneal transplant is cross-linked to render thecorneal tissue more resilient to expansion as occurs after refractivesurgery.

In one embodiment, the photosensitizer (e.g., riboflavin) or othercrosslinkers are conjugated with cell penetrating peptides (CPP) oractivatable cell penetrating peptides (ACPP) or other cell penetratingagents, such as cyclodextrin, etc. that enhances penetration of thecrosslinker in the tissue, saving time for surgery if the crosslinkingis done on a patient, or if the crosslinking is done for the storage ofthe tissue.

In one embodiment, the photosensitizer formulation permits fasterpenetration of the crosslinker inside the corneal transplant, thusreducing the operative time less than ½, ⅓, ⅕ or less than 1/10 of thetime that it would take to cross-link the corneal tissue as done by thestandard procedure.

In one embodiment, the crosslinkers are made in the form ofnanoparticles, such as dendrimers, and can be organic, non-organic,metallic, such as gold, iron or ferric oxide or zinc, silver, silica,graphene, carbon nanotubes, nanowires, quantum dots (QDs), etc. orcoated with lipids, or an antibody that encourages the cell penetration,etc.

In one embodiment, the crosslinker is conjugated with CPP and antibodyagainst a bacterium, fungi, viruses, prions, parasites, attached toeither metallic or organic nanoparticles, such as a carbon nanospheresor nanotubes, or gold or silver or zinc nanoparticles, etc. and CPP, andradiated with UV light or other wavelengths of light that is absorbed bythe nanoparticles and the photosensitizer heats up thenanoparticle/pathogens complex creating thermal damage to the pathogensin addition to being cross-linked by the photosensitizer. The use of UVradiation, in addition, damages the genetic components of the pathogensalong with the medication that kills the pathogens or makes them moresensitive to the medication to which the pathogens would not besensitive normally or were resistant, such as methicillin-resistantstaphylococcus aureus (MRSA) bacteria, after administering thecrosslinker complex locally or topically.

In one embodiment, the antibody-coated nanoparticles can be attached tothe antimicrobial peptides, and the crosslinker is applied topically tothe eye, lid, skin or mucosa, or any reachable infection even with afiber optic (e.g., in the lung, eye, bladder, vagina, uterus, throat,nose, or exposed tumor) as needed and radiated with UV light tocrosslink the protein of the bacteria and kill them along withpotentially a cancerous lesion or cells.

In one embodiment, the process of crosslinking using UV radiationcreates singlet oxygen and reactive oxygen species in the presence ofthe oxygen that crosslinks the proteins, including those present in thebacteria, fungi, viruses, prions, and parasites, thereby killing them,and in case of the cornea or the sclera, crosslinks the collagens in thetissue and makes then less immunogenic.

In one embodiment, the corneal tissue is cross-linked to be used inrefractive surgery as a corneal inlay to change the refractive power ofthe cornea. In another embodiment, it replaces the damaged cornealtissue. In yet another embodiment, the cross-linked cornea or sclera canbe used as a tissue filler or provide a shape for the surrounding tissuestructures, such as in cosmetic surgery in the face, replacement for thedamaged lids, reconstructing the facial appearance, etc. In stillanother embodiment, it can serve as a protecting container to preventimmune response to the cells, etc. inside the container or can be usedas a reservoir for drug delivery etc.

In one embodiment, the cross-linked cornea or sclera is co-cultured in amedium having nanoparticles/CPP or lipids that penetrates the tissuesscaffold, and the nanoparticles are made of polylactic, polyglycolic orpolycaprolactone of silicon nanoparticles conjugated with one or moremedications (e.g., anti-inflammatory agents, steroids, NSAIDs,anti-proliferative agents, immunosuppressives, such as cyclosporine,mycophenolic acid, antibiotics, anti-glaucoma agents, such asprostaglandin analogues, Rock inhibitors, such as Fasudil, rhopressa,etc., anti-integrins, Wnt inhibitors, or GSK inhibitors, anti-VEGFs, orcombinations thereof). The cross-linked cornea or sclera can be cut toany size or thickness (e.g., 10 microns to 1 millimeter and more) beradius shaped, circular, rectangular, etc. to release the medicationdepending on the bio-degradation of polymeric nanoparticles. The implantcan be implanted anywhere in the eye (see e.g., FIG. 4E).

In one embodiment, the cross-linked cornea or sclera is made any desiredshape and thickness using a femtosecond laser or an excimer laser, andusing the same lasers one can punch partial or full thickness holes inthe implant, so that after penetration of the nanoparticles, the releasetime will depend on the number and the size of the holes created insidethe cross-linked cornea ranging from 1 month to few years, thecross-linked cornea or sclera is then co-cultured in a medium havingnanoparticles/CPP or lipids that penetrate the tissues scaffold andnanoparticles are made of polylactic, polyglycolic or polycaprolactoneof silicon nanoparticles conjugated with medications (e.g.,anti-inflammatory agents, steroids, NSAIDs, anti-proliferative agents,immunosuppressives, such as cyclosporine, mycophenolic acid,antibiotics, anti-glaucoma agents, such as prostaglandin analogues, Rockinhibitors, such as Fasudil, rhopressa, etc., anti-integrins, Wntinhibitors, or GSK inhibitors, anti-VEGFs, or a combination thereof).The cross-linked cornea or sclera can be cut to any size or thickness(e.g., 10 microns to 1 millimeter and more), be radius shaped, circular,rectangular, etc. to release the medication depending on thebio-degradation of polymeric nanoparticles. The implant can be implantedanywhere in the eye or enhanced its release by a focused ultrasound.

In one embodiment, the scleral tissue is cross-linked for long termstorage.

In one embodiment, the photosensitizer, such as riboflavin, is in aphysiological solution with osmolarities of 200 mOsm/L to 400 mOsm/L, orpreferably about 300 mOsm/L.

In one embodiment, the pH of the solution is between 6-8 pH orpreferably 7.4 pH.

In one embodiment, the photosensitizer solution contains dextran of 0.1%-3%.

In one embodiment, the photosensitizer/nanoparticle solution can containlow molecular weight heparin or hyaluronic acid or chondroitin sulfate,etc., the low molecular weight heparin enhances the killing of thebacteria in vitro and in vivo after topical application and irradiationwith UV light or other light. These preparations can be used fortreatment of bacterial, fungi, viral, parasitic keratitis orconjunctivitis.

The crosslinking of the tissue is done when the photosensitizer isradiated with a light wavelength of ultraviolet to infrared depending onthe photosensitizer that absorbs the light beam. In one embodiment, thephotosensitizer solution can, in addition, contain some additives, suchas antibiotics, antifungals, antivirals, and/or anti-parasites.

In one embodiment, the corneal crosslinker is conjugated with CPP,nanoparticles applied to the cornea and irradiated with UV light orother light to kill all cellular elements of the cornea and sclera andcrosslink the collagen, and sterilize the corneal implant for storageprior to its use.

In one embodiment, for the cross-linked corneal storage (CCS), thecorneal crosslinker is conjugated with CPP, antibody-coated metallicnanoparticles, such as gold nanoparticles, etc. When the implant iscompletely penetrated by the crosslinker, it is radiated with UV lightor other light wavelengths to kill the bacteria by thermal energy, andthe crosslinking kills bacteria, fungi, parasites, viruses and prionsfor corneal storage prior to its use.

In still a further embodiment, the photosensitizer is conjugated withone or more metallic or non-metallic nanoparticles and/or one or moreantibody-coated nanoparticles, and the one or more nanoparticles and/orone or more antibody-coated nanoparticles are further conjugated withone or more cell penetrating peptides (CPP) and/or activatable-cellpenetrating peptides (ACPP), thereby forming aphotosensitizer-nanoparticle-CPP/ACPP complex for facilitating anenhanced penetration of the photosensitizer into a corneal stroma andstromal cells of the host cornea with or without the use of lowintensity ultrasound to kill the stromal cells after UV radiation.

In a further embodiment of the present invention, the step ofcross-linking the corneal implant prior to implantation of the corneacomprises the substeps of: (a) applying a photosensitizer to the corneaso as to penetrate the entire corneal implant; (b) irradiating thecornea with ultraviolet light so as to activate cross-linkers in thecornea and thereby crosslink the entire corneal implant; (c) cutting thecross-linked cornea to the desired shape or dioptric power; (d) creatinga pocket in the recipient cornea; and (e) implanting the cross-linkedcornea inside the corneal pocket along with a lubricating substance,such as hyaluronic acid, etc.

In one embodiment, the corneal inlay is implanted as above, thencross-linked with a photosensitizer injected inside the corneal pocketto penetrate the implant and the wall of the corneal pocket, and thenthe cornea and the inlay are simultaneously cross-linked. Followed bycopious washing of the cornea with saline and/or an anti-inflammatoryagent, such as steroid, NSAIDs, or Rock inhibitors, anti-integrins, orGSK inhibitors or Wnt inhibitors.

In still a further embodiment, the photosensitizer is conjugated withone or more nanoparticles and/or one or more antibody-coatednanoparticles, and the one or more nanoparticles and/or one or moreantibody-coated nanoparticles are further conjugated with anantimicrobial peptide that damages the bacterial cell membrane while thenormal cells are not affected, and one or more cell penetrating peptides(CPP) and/or activatable-cell penetrating peptides (ACPP), and therebyforming a photosensitizer-nanoparticle-CPP/ACPP complex for facilitatingan enhanced penetration of the photosensitizer into a corneal stroma andstromal cells of the host cornea to kill the stromal cells. Thepreparation is administered inside an infected corneal pocket of a largeabscess, of nasolacrimal duct, or the lid, or skin or mucosa andirradiated with the light to activate the photosensitizer and kill allorganisms while protecting the normal tissue.

In still a further embodiment, the photosensitizer is conjugated withone or more antibody-coated nanoparticles or dendrimers, and the one ormore antibody-coated nanoparticles or dendrimers are further conjugatedwith one or more cell penetrating peptides (CPP) and/or activatable-cellpenetrating peptides (ACPP), thereby forming aphotosensitizer-antibody-coated nanoparticle or dendrimer-CPP/ACPPcomplex; and the method further comprises the steps of: (iv)administering the photosensitizer-antibody-coated nanoparticle ordendrimer-CPP/ACPP complex topically or by injection to the host cornea,the photosensitizer being absorbed by bacteria, viruses, fungi, and/orprotozoa present in an infected corneal ulcer of the host cornea; and(v) irradiating the host cornea with light so as to kill the bacteria,viruses, fungi, or protozoa by cross-linking the cytoplasmic proteins ofthe bacteria, viruses, fungi, and/or protozoa by thermal energy andcrosslinking of the proteins of the micro-organisms, viruses, and/orprions, and sterilizing the entire cross-linked corneal and scleralimplant's thickness and killing all pathogens and cells in the implantand cleansed with a physiological solution to wash out the toxins andthe debris in the tissue.

In one embodiment, the antimicrobial peptides are from the group ofchemotactic peptides having inflammatory and antimicrobial activity,such as polybia-CP (Pol-CP-NH₂:Ile-Leu-Gly-Thr-Ile-Leu-Gly-Leu-Leu-Lys-Ser-Leu-NH₂), Pol-CP-NH₂, andprotonectin and polybia-MPI, linear cationic amphipathic AMPs, or waspPolybia paulista.

In one embodiment, when the patient suffers from an infected cornealulcer, or skin ulcer, or mucosa ulcer, the crosslinker is conjugatedwith CPP, antibiotics, antifungals, anti-parasites, antivirals etc. andthe antibody-coated metallic nanoparticles, such as gold nanoparticles,and the ulcer is treated with a topical application of the crosslinkercomplex to the ulcer, followed by UV radiation or other lightwavelengths to kill the bacteria/fungi parasites, or viruses, etc. bythermal energy, medication, and crosslinking the proteins of thebacteria, fungi, parasites, viruses, while crosslinking the cornea orthe skin to resist enzymatic degradation and healing the infected ulcer.

In one embodiment, corneal crosslinking is performed with CPP,antibody-coated metallic gold nanoparticles and antibiotics and/orantimicrobial proteins, etc. using UV or other light wavelengths to killthe bacteria by thermal energy and crosslinking. The thermal energy andcrosslinking kills bacteria, fungi, parasites, viruses and prions, andtheir infectious pathogens, such as viral infections, such as hepatitisB, West Nile virus, Chagas disease, and prion agents.

In one embodiment, scleral crosslinking with CPP, nanoparticles using UVlight or other wavelengths of light kills all cellular elements of thecornea and/or sclera, and crosslinks the collagen, and sterilizes theimplant.

In one embodiment, scleral crosslinking with CPP, antibody-coatedmetallic gold nanoparticles, etc. using UV light or other lightwavelengths kills the bacteria, fungi, parasites, viruses and prions bythermal energy and crosslinking.

In one embodiment, scleral crosslinking with CPP, antibody-coatedmetallic gold nanoparticles and antibiotics and/or antimicrobialproteins, etc. using UV or other light wavelengths kills the bacteria,fungi, parasites, viruses and prions by thermal energy and crosslinking,and subsequently, the tissue is washed with a physiologic solution andexposed to low frequency ultrasound to cleanse the tissue toxinsreleased by killing the bacteria, fungi, viruses, prions, or parasites.

In one embodiment, after storage of the cross-linked cornea or sclera,if the cross-linked tissue appears swollen, it can be treated with aphysiological solution containing antibiotics, deturgescent agents likedextran 4-8%, and chondroitin sulphate, resulting in deswelling of thecornea or the sclera.

In one embodiment, the cross-linked tissue after long term storage canbe re-sterilized with gamma radiation.

In one embodiment, the corneas can be stored using standard techniqueswhile suspended in 80 ml Eagle's MEM containing HEPES buffer, 26 mmol/lNaHCO₃, 2% FBS, 2 mmol/l L-glutamine, penicillin, streptomycin andamphotericin B, and stored at 34° C. for 1-7 days, then, if they havenot used for corneal transplantation, they can now prepared as describedabove for crosslinking or the cornea and sclera, which can be thenstored for greater than 1 year, etc.

In one embodiment, the cross-linked cornea is denuded from cornealepithelial cells and endothelial cells while keeping the Descemet'smembrane co-cultured with stem cells, or endothelial stem cells, orpre-corneal conjunctival stem cells, or mesenchymal stem cells of thepotentially host patient with polymeric anti-inflammatory nanoparticlesof PLA, PLA GLA having Rock inhibitors, anti-integrins, GSK inhibitors,and/or immunosuppressive agents combined with electrophoresis tissuepenetration in the implant and/or followed with medical treatment usingtopical antibiotics, anti-inflammatory compounds, such as Rockinhibitors, anti-integrins, GSK inhibitors, and/or immunosuppressiveagents.

In one embodiment, the cross-linked cornea is denuded from cornealepithelial cells and endothelial cells while keeping the Descemet'smembrane co-cultured with stem cells, or endothelial stem cells, orpre-corneal conjunctival stem cells, or mesenchymal stem cells of thepotentially host patient and polymeric nanoparticles conjugated withanti-inflammatory compounds, such as Rock inhibitors, anti-integrins,GSK inhibitors, and/or immunosuppressive agents, such as cyclosporine,mycophenolic acid, tacrolimus, combined with electrophoresis tissuepenetration in the cross-linked implant etc. to release the medicationafter implantation for a long time after implantation.

In one embodiment, the cross-linked cornea is denuded from cornealepithelial cells and endothelial cells while keeping the Descemet'smembrane co-cultured with stem cells, endothelial stem cells, andtransfection with transcription factors such as E2F2, which has beenreported to stimulate cell-cycle progression and endothelial cellreplication to grow endothelial cells on the Descemet's membrane fortransplantation in a patient. The technology can be used as Descemetstripping automated endothelial keratoplasty (DSEK) or Descementmembrane endothelial keratoplasy (DMEK) or deep lamellar keratoplasty(DLK) for partial transplantation of the cornea that is now cross-linkedwith or without corneal endothelial cells as known in the art,simplifying the 3-D printing of the cornea, for the transplantation,since one has now a resilient cross-linked cornea to work with, and theprocess can be made simple by manufacturing all the tissues except forgrowing endothelial cells on the back of a cross-linked cornea with itsDescemet's membrane intact, thus reducing the chance of the rejectionsince the corneal stroma part of the transplant is cross-linked and willnot induce an immune response in the host followed after implantationwith topical or subconjunctival medical treatment using antibiotics,anti-inflammatory compounds, such as Rock inhibitors, anti-integrins,GSK inhibitors, and/or immunosuppressive agents.

In one embodiment, one can use a combination or cross-linked cornea orcross-linked animal cornea or genetically engineered animal cornea togrow corneal endothelial cells prior to their implantation in DSEK orDMEK, since the cross-linked corneal stromas are not immunogenic. Thecross-linked tissue is co-cultured with a medium havingnanoparticles/CPP or lipids that penetrates the tissue scaffolds and thenanoparticles are made of polylactic, polyglycolic or polycaprolactoneor silicon nanoparticles conjugated with medications, such asanti-inflammatory agents, steroids, NSAID, anti-proliferative agents,immunosuppressives, such as cyclosporine, mycophenolic acid,antibiotics, anti-glaucoma agents, such as prostaglandin analogues, Rockinhibitors, such as Fasudil, rhopressa, etc., anti-integrins, Wntinhibitors, or GSK inhibitors, anti-VEGFs, or combinations thereofcombined with or without electrophoresis tissue penetration in theimplant thereof, to release the medication after implantation dependingon the bio-degradation of polymeric nanoparticles.

In one embodiment, one can use a combination or cross-linked cornea orcross-linked animal cornea or genetically engineered animal cornea togrow corneal endothelial cells prior to their implantation in DSEK orDMEK since the cross-linked corneal stromas are not immunogenic. Thecross-linked tissue is co-cultured with a medium havingnanoparticles/CPP or lipids that penetrates the tissue scaffolds and thenanoparticles are made of polylactic, polyglycolic or polycaprolactoneor silicon nanoparticles conjugated with medications, such asanti-inflammatory agents, steroids, NSAIDs, anti-proliferative agents,immunosuppressives, such as cyclosporine, mycophenolic acid,antibiotics, anti-glaucoma agents, such as prostaglandin analogues, Rockinhibitors, such as Fasudil, rhopressa, etc., anti-integrins, Wntinhibitors, or GSK inhibitors, anti-VEGFs, or combinations thereof andthe tissue is exposed to low intensity ultrasound to enhance penetrationof the nanoparticles in the implant and to release the medication afterimplantation slowly, depending on the bio-degradation of polymericnanoparticles to release the medication after implantation depending onthe bio-degradation of polymeric nanoparticles.

In one embodiment, one can use a combination or cross-linked cornea orcross-linked animal cornea or genetically engineered animal cornea togrow corneal endothelial cells prior to their implantation in DSEK orDMEK since the cross-linked corneal stromas are not immunogenic. Thecross-linked tissue is co-cultured with a medium havingnanoparticles/CPP or lipids that penetrates the tissue scaffolds andnanoparticles are made of polylactic, polyglycolic or polycaprolactoneor silicon nanoparticles conjugated with medications, such asanti-inflammatory agents, steroids, NSAIDs, anti-proliferative agents,immunosuppressives, such as cyclosporine, mycophenolic acid,antibiotics, anti-glaucoma agents, such as prostaglandin analogues, Rockinhibitors, such as Fasudil, rhopressa, etc., anti-integrins, Wntinhibitors, or GSK inhibitors, anti-VEGFs, or their combination, and thetissue is exposed to low intensity ultrasound to enhance penetration ofthe nanoparticles in the implant and to release the medication afterimplantation slowly, depending on the bio-degradation of polymericnanoparticles.

In one embodiment, the cross-linked cornea can be re-cellularized byeither injecting appropriate stem cells inside the cross-linked implant,or after implantation, encouraging stem cells, such as mesenchymal cellsmigration from the adjacent tissue. In either case, the topicaladministration of nerve growth factors alone or Rock inhibitors alone,or a combination of them encourage cell migration and migration of nerveazons in the cross-linked graft.

In one embodiment, the epithelial cells can be seeded on thecross-linked Bowman's membrane or the endothelial cells seeded on theDescemet's membrane prior or after implantation.

In one embodiment, the corneal or scleral graft can be built using astandard 3-D printing technique using the cross-linked cornea or a partof it as a base in addition to use of one or the other polymericmaterials such as Silk sericin, Poly(vinyl alcohol), Poly(2-hydroxyethylmethacrylate), Polyethylene (glycol) diacrylate, Poly(lactic-co-glycolicacid), Poly(ethylene glycol), Poly(acrylic acid), Poly(glycerolsebacate), Poly(ester urethane) urea, Peptide amphiphiles,naturally/synthetically derived, m Arginine-glycine-aspartic acid, thecellular elements of the cornea can be added subsequently in the tissueculture, such a hybrid cornea can renders itself to modification of itsrefractive power after implantation or before implantation, using lowenergy level pulses of a femtosecond laser.

Any of the features, attributes, or steps of the above describedembodiments and variations can be used in combination with any of theother features, attributes, and steps of the above described embodimentsand variations as desired.

Although the invention has been shown and described with respect to acertain embodiment or embodiments, it is apparent that this inventioncan be embodied in many different forms and that many othermodifications and variations are possible without departing from thespirit and scope of this invention.

Moreover, while exemplary embodiments have been described herein, one ofordinary skill in the art will readily appreciate that the exemplaryembodiments set forth above are merely illustrative in nature and shouldnot be construed as to limit the claims in any manner. Rather, the scopeof the invention is defined only by the appended claims and theirequivalents, and not, by the preceding description.

The invention claimed is:
 1. A method of corneal and scleral inlaycrosslinking and preservation, the method comprising the steps of:cross-linking at least a portion of a donor cornea or a donor sclera soas to kill cellular elements in the portion of the donor cornea or thedonor sclera, and make the portion of the donor cornea or the donorsclera less antigenic to a body portion of a recipient patient in whichthe portion of the donor cornea or the donor sclera is to be implanted;and storing the cross-linked donor cornea or the donor sclera for a longperiod of time prior to implanting the portion of the donor cornea orthe donor sclera into the body portion of the recipient patient.
 2. Themethod according to claim 1, wherein the step of cross-linking theportion of the donor cornea or the donor sclera comprises the substepsof: applying a photosensitizer to the portion of the donor cornea or thedonor sclera so as to penetrate the tissue of the donor cornea or thedonor sclera; and irradiating the portion of the donor cornea or thedonor sclera with ultraviolet light so as to cross-link collagen in theportion of the donor cornea or the donor sclera so as to make theportion of the donor cornea or the donor sclera less antigenic to thebody portion of the recipient patient in which the portion of the donorcornea or the donor sclera is to be implanted.
 3. The method accordingto claim 2, wherein the photosensitizer comprises riboflavin and a cellpenetrating agent, and wherein the portion of the donor cornea or thedonor sclera is irradiated by using a laser emitting the ultravioletlight.
 4. The method according to claim 3, wherein the cell penetratingagent comprises one or more nanoparticles conjugated with one or morecell penetrating peptides (CPP) and/or activatable-cell penetratingpeptides (ACPP).
 5. The method according to claim 4, wherein after thesubstep of irradiating the portion of the donor cornea or the donorsclera with ultraviolet light, the method further comprises the stepsof: implanting the portion of the donor cornea into a host cornea of therecipient patient; and applying low intensity focused ultrasound tostromal cells of the host cornea of the recipient patient so as to killthe stromal cells of the host cornea.
 6. The method according to claim1, wherein the step of storing the cross-linked donor cornea or thedonor sclera for a long period of time comprises storing thecross-linked donor cornea or the donor sclera for a time period of oneyear or more.
 7. The method according to claim 1, wherein, prior to thestep of cross-linking at least a portion of the donor cornea or thedonor sclera, the method comprises the steps of: dissecting and removingall of the soft tissue from a donor eye so to leave only the donorcornea and/or the donor sclera, the removed soft tissue of the donor eyecomprising the conjunctiva, muscles, retina, choroid, iris, ciliarybody, and optic nerve; and cleansing the donor cornea and/or the donorsclera in a physiological solution so as to prepare the donor corneaand/or the donor sclera for cross-linking.
 8. The method according toclaim 1, further comprising the step of: cutting and shaping the donorcornea using a cutting instrument so as to form a lens having a desiredthickness, shape, and diameter; the lens comprising a plus or minuslens, an astigmatic lens, or a combination thereof.
 9. The methodaccording to claim 1, wherein, prior to the step of cross-linking atleast a portion of the donor cornea or the donor sclera, the methodcomprises the step of: exposing the donor cornea or the donor sclera toa high intensity ultrasonic or hydrostatic force so as to destroy thecellular elements in the portion of the donor cornea or the donorsclera; and wherein, after the step of cross-linking at least a portionof the donor cornea or the donor sclera, the method comprises the stepof: cleansing the donor cornea and/or the donor sclera in a salinesolution to remove the reactive oxygen species and enzymes released fromthe destroyed cellular elements by mechanical agitation of the donorcornea or the donor sclera or by applying a low frequency ultrasound tothe donor cornea or the donor sclera.
 10. The method according to claim1, wherein, after the step of cross-linking at least a portion of thedonor cornea or the donor sclera, the method comprises the step of:implanting the portion of the donor cornea or the donor sclera in thebody of the recipient patient to replace an injured chin, lip, nose, orcheek; or in cosmetic surgery, to augment a chin, lip, nose, or cheek.11. The method according to claim 1, wherein the step of cross-linkingthe portion of the donor cornea or the donor sclera comprises thesubsteps of: applying a photosensitizer comprising riboflavin to thedonor cornea or the donor sclera so as to penetrate the tissue of thedonor cornea or the donor sclera; and irradiating the donor cornea orthe donor sclera with ultraviolet light so as to sterilize the donorcornea or the donor sclera.
 12. The method according to claim 1, whereinthe recipient patient has an infected corneal ulcer or scleral weaknessor infection, and wherein the method further comprises the steps of:applying a photosensitizer conjugated with one or more antibody-coatedmetallic nanoparticles, one or more cell penetrating peptides (CPP), andone or more medications to a host eye of the recipient patient, the oneor more medications selected from the group consisting of antibiotics,antifungals, anti-parasites, antivirals, and combinations thereof; andirradiating the area of the host eye having the infected corneal ulceror the scleral weakness or infection with ultraviolet light so as tokill bacteria, fungi, parasites, and/or viruses while cross-linking thearea of the host eye to resist enzymatic degradation and heal theinfected corneal ulcer or the scleral weakness or infection.
 13. Themethod according to claim 1, wherein the method further comprises thesteps of: forming cavities or holes in the portion of the cross-linkeddonor cornea or the donor sclera using a laser so that the portion ofthe donor cornea or the donor sclera is capable of being used forsustained drug delivery; and co-culturing the donor cornea or the donorsclera in a medium having nanoparticles conjugated with one or moremedications and cell penetrating peptides (CPP) or lipids that penetratethe tissue scaffolds formed by the cavities or holes in the portion ofthe donor cornea or the donor sclera, the portion of the donor cornea orthe donor sclera configured to be used for sustained drug delivery whenthe portion of the donor cornea or the donor sclera is implanted in thebody portion of the recipient patient.
 14. The method according to claim13, wherein the nanoparticles are formed from polylactic, polyglycolic,polycaprolactone, and/or silicon; and wherein the one or moremedications are selected from the group consisting of anti-inflammatoryagents, steroids, nonsteroidal anti-inflammatory drugs (NSAIDs),anti-proliferative agents, immunosuppressives, antibiotics,anti-glaucoma agents, Rock inhibitors, anti-integrins, Wnt inhibitors,GSK inhibitors, anti-vascular endothelial growth factors (anti-VEGFs),and combinations thereof.
 15. The method according to claim 1, whereinthe step of cross-linking the portion of the donor cornea or the donorsclera comprises the substeps of: applying riboflavin to the portion ofthe donor cornea or the donor sclera so as to penetrate the tissue ofthe donor cornea or the donor sclera; and irradiating the portion of thedonor cornea or the donor sclera using a laser emitting ultravioletlight so as to cross-link collagen in the portion of the donor cornea orthe donor sclera so as to make the portion of the donor cornea or thedonor sclera less antigenic to the body portion of the recipient patientin which the portion of the donor cornea or the donor sclera is to beimplanted.
 16. The method according to claim 15, wherein, prior to thestep of cross-linking the portion of the donor cornea or the donorsclera with the riboflavin and the ultraviolet light, the methodcomprises the step of: using chemical means or mechanical means todestroy the cellular elements in the portion of the donor cornea or thedonor sclera.
 17. The method according to claim 16, wherein the chemicalmeans for destroying the cellular elements in the portion of the donorcornea or the donor sclera are selected from the group consisting ofethanol, glycerol, acids, alkalis, peracetic acid, ammonium hydroxideionic detergents, sodium dodecyl sulfate, sodium deoxycholate non-ionicdetergents, zwitterionic detergents, Triton X-100, benzalkoniumchloride, Igepal, genipin, and combinations thereof.
 18. The methodaccording to claim 16, wherein the mechanical means for destroying thecellular elements in the portion of the donor cornea or the donor scleracomprises applying low intensity focused ultrasound to the portion ofthe donor cornea or the donor sclera.